U.S. patent application number 10/363082 was filed with the patent office on 2004-02-12 for packaging of positive-strand rna virus replicon particles.
Invention is credited to Gangolli, Seema, Kovacs, Gerald R., Kowalski, Jacek, Vasilakis, Nikos, Zamb, Timothy.
Application Number | 20040029279 10/363082 |
Document ID | / |
Family ID | 22859031 |
Filed Date | 2004-02-12 |
United States Patent
Application |
20040029279 |
Kind Code |
A1 |
Kovacs, Gerald R. ; et
al. |
February 12, 2004 |
Packaging of positive-strand rna virus replicon particles
Abstract
The invention generally relates to recombinant polynucleotides,
positive-strand RNA virus (psRNAV) recombinant expression vectors,
and packaging systems. The packaging systems are based on the
expression of helper functions by coinfecting re-combinant poxvirus
vectors comprising recombinant polynucleotides. Methods for
obtaining psRNAV replicon particles using these packaging systems
are disclosed. Immunogenic compositions and pharmaceutical
formulations are provided that comprise replicon particles of the
invention. Methods for generating an immune response or producing a
pharmaceutical effect are also provided.
Inventors: |
Kovacs, Gerald R.;
(Rockville, MD) ; Vasilakis, Nikos; (Galveston,
TX) ; Kowalski, Jacek; (Mahwah, NJ) ;
Gangolli, Seema; (Park Ridge, NJ) ; Zamb,
Timothy; (Nyack, NY) |
Correspondence
Address: |
WYETH
PATENT LAW GROUP
FIVE GIRALDA FARMS
MADISON
NJ
07940
US
|
Family ID: |
22859031 |
Appl. No.: |
10/363082 |
Filed: |
August 27, 2003 |
PCT Filed: |
August 28, 2001 |
PCT NO: |
PCT/US01/41888 |
Current U.S.
Class: |
435/456 ;
435/235.1; 536/23.72 |
Current CPC
Class: |
C07H 21/04 20130101;
C12N 2710/24144 20130101; C12N 2770/36152 20130101; C12N 2770/36143
20130101; A61P 35/00 20180101; A61P 37/08 20180101; C12N 2710/24143
20130101; A61P 31/00 20180101; C12N 2830/002 20130101; A61K 48/00
20130101; A61K 2039/5256 20130101; C12N 15/86 20130101; A61P 25/28
20180101; A61K 38/1709 20130101 |
Class at
Publication: |
435/456 ;
435/235.1; 536/23.72 |
International
Class: |
C12N 015/86; C07H
021/04; C12N 007/00 |
Claims
What is claimed is:
1. A recombinant polynucleotide comprising: a first portion
comprising a sequence encoding a DNA-dependent RNA polymerase
operatively linked to a first heterologous promoter; and a second
portion comprising a sequence encoding at least one positive-strand
RNA virus (psRNAV) structural protein, but not all of the psRNAV
structural proteins, operatively linked to a second heterologous
promoter.
2. The recombinant polynucleotide of claim 1, wherein the
DNA-dependent RNA polymerase of the first portion is selected from
T3, T7, and SP6 DNA-dependent RNA polymerase; wherein the first
heterologous promoter is a poxvirus promoter; wherein the at least
one psRNAV structural protein of the second portion is an
alphavirus structural protein selected from an alphavirus capsid
and an alphavirus glycoprotein; and wherein the second heterologous
promoter binds to said DNA-dependent RNA polymerase.
3. The recombinant polynucleotide of claim 2, wherein the
DNA-dependent RNA polymerase of the first portion is a T7
polymerase; wherein the poxvirus promoter is a vaccinia virus
synthetic early/late promoter; wherein the second heterologous
promoter binds to a T7 DNA-dependent RNA polymerase; and wherein
the alphavirus capsid is a Venezuelan equine encephalitis virus
(VEE) capsid and the alphavirus glycoprotein is a VEE
glycoprotein.
4. The recombinant polynucleotide of claim 1, wherein the at least
one psRNAV structural protein of the second portion is selected
from an alphavirus structural protein, a rubella virus structural
protein, a coronavirus structural protein, a dengue virus
structural protein, and a Hepatitis C virus structural protein.
5. A recombinant polynucleotide comprising: a first portion
comprising a sequence encoding at least one psRNAV structural
protein, but not all of the psRNAV structural proteins, operatively
linked to a first heterologous promoter; and a second portion
comprising a second heterologous promoter operatively linked to a
psRNAV replicon comprising an psRNAV subgenomic promoter
operatively linked to a sequence encoding at least one foreign
polypeptide.
6. The recombinant polynucleotide of claim 5, wherein the at least
one psRNAV structural protein is an alphavirus structural protein
selected from an alphavirus capsid and an alphavirus glycoprotein;
and wherein the first and second heterologous promoters both bind
to a polymerase selected from T3, T7, and SP6 DNA-dependent RNA
polymerase.
7. The recombinant polynucleotide of claim 6, wherein the
alphavirus capsid is a VEE capsid and the alphavirus glycoprotein
is a VEE glycoprotein; and wherein the first and second
heterologous promoters both bind to a T7 DNA-dependent RNA
polymerase.
8. The recombinant polynucleotide of claim 5, wherein the at least
one psRNAV structural protein of the first portion is selected from
an alphavirus structural protein, a rubella virus structural
protein, a coronavirus structural protein, a dengue virus
structural protein, and a Hepatitis C virus structural protein; and
the psRNAV replicon of the second portion is selected from an
alphavirus replicon, a rubella virus replicon, a coronavirus
replicon, a dengue virus replicon, and a Hepatitis C virus
replicon.
9. A recombinant polynucleotide comprising: a first portion
comprising a sequence. encoding a DNA-dependent RNA polymerase
operatively linked to a first heterologous promoter; and a second
portion comprising a sequence encoding a replicon-like psRNAV
helper RNA sequence operatively linked to a second heterologous
promoter.
10. The recombinant polynucleotide of claim 9, wherein the
DNA-dependent RNA polymerase of the first portion is selected from
a T3, T7, and SP6 DNA-dependent RNA polymerase; wherein the first
heterologous promoter is a poxvirus promoter, wherein the
replicon-like psRNAV helper RNA sequence of the second portion is
an alphavirus helper RNA sequence comprising a sequence encoding an
alphavirus structural protein selected from an alphavirus capsid
and an alphavirus glycoprotein; and wherein the second heterologous
promoter binds to apolymerase selected from T3, T7, and SP6
DNA-dependent RNA polymerase.
11. The recombinant polynucleotide of claim 10, wherein the
DNA-dependent RNA polymerase of the first portion is a T7
DNA-dependent RNA polymerase; wherein the poxvirus promoter is a
vaccinia virus synthetic early/late promoter; wherein the
alphavirus capsid is a VEE capsid and the alphavirus glycoprotein
is a VEE glycoprotein; and wherein the second heterologous promoter
binds to a T7 DNA-dependent RNA polymerase.
12. The recombinant polynucleotide of claim 9, wherein the psRNAV
helper RNA sequence is selected from an alphavirus helper RNA
sequence, a rubella virus helper RNA sequence, a coronavirus helper
RNA sequence, a dengue virus helper RNA sequence, and a Hepatitis C
virus helper RNA sequence.
13. A recombinant polynucleotide comprising: a first portion
comprising a sequence encoding a replicon-like psRNAV helper RNA
sequence operatively linked to a first heterologous promoter; and a
second portion comprising a second heterologous promoter
operatively linked to a psRNAV replicon comprising a psRNAV
subgenomic promoter operatively linked to a sequence encoding at
least one foreign polypeptide.
14. The recombinant polynucleotide of claim 13, wherein the
replicon-like psRNAV helper RNA sequence of the first portion is an
alphavirus helper RNA sequence comprising a sequence encoding an
alphavirus structural protein selected from an alphavirus
glycoprotein and an alphavirus capsid; and wherein the first and
second heterologous promoters both bind to a polymerase selected
from T3, T7, and SP6 DNA-dependent RNA polymerase.
15. The recombinant polynucleotide of claim 14, wherein the
alphavirus capsid is a VEE capsid and the alphavirus glycoprotein
is a VEE glycoprotein; and wherein the first and second promoters
both bind to a T7 DNA-dependent RNA polymerase.
16. The recombinant polynucleotide of claim 13, wherein the psRNAV
helper RNA sequence of the first portion is selected from an
alphavirus helper RNA sequence, a rubella virus helper RNA
sequence, a coronavirus helper RNA sequence, a dengue virus helper
RNA sequence, and a Hepatitis C virus helper RNA sequence; and the
psRNAV replicon of the second portion is selected from an
alphavirus replicon, a rubella virus replicon, a coronavirus
replicon, a dengue virus replicon, and a Hepatitis C virus
replicon.
17. A recombinant vector comprising a viral vector and the
recombinant polynucleotide of any of claims 1-16.
18. A recombinant modified vaccinia virus Ankara (MVA) comprising
the recombinant polynucleotide of any of claims 1-16.
19. A psRNAV replicon packaging system comprising: (a) a
recombinant MVA comprising the recombinant polynucleotide of claim
1, wherein the at least one psRNAV structural protein of the second
portion is selected from a psRNAV capsid and a psRNAV glycoprotein;
and (b) a recombinant MVA comprising the recombinant polynucleotide
of claim 5, wherein the at least one psRNAV structural protein of
the first portion is selected from a psRNAV capsid and a psRNAV
glycoprotein; wherein the psRNAV structural protein of (a) and the
psRNAV structural protein of (b) are not the same.
20. A psRNAV replicon packaging system comprising: (a) a
recombinant MVA comprising the recombinant polynucleotide of claim
9, wherein the replicon-like psRNAV helper RNA sequence of the
second portion is a sequence encoding a psRNAV structural protein
selected from a psRNAV capsid and a psRNAV glycoprotein; and (b) a
recombinant MVA comprising the recombinant polynucleotide of claim
13, wherein the replicon-like psRNAV helper RNA sequence of the
first portion is a sequence encoding a psRNAV structural protein
selected from a psRNAV capsid and a psRNAV glycoprotein; wherein
the psRNAV structural protein of (a) and the psRNAV structural
protein of (b) are not the same.
21. An alphavirus replicon packaging system comprising: (a) a
recombinant MVA comprising the recombinant polynucleotide of claim
3; and (b) a recombinant MVA comprising the recombinant
polynucleotide of claim 7; wherein the alphavirus structural
protein of (a) and the alphavirus structural protein of (b) are not
the same.
22. An alphavirus replicon packaging system comprising: (a) a
recombinant MVA comprising the recombinant polynucleotide of claim
11; and (b) a recombinant MVA comprising the recombinant
polynucleotide of claim 15; wherein the alphavirus structural
protein of (a) and the alphavirus structural protein of (b) are not
the same.
23. A psRNAV replicon packaging system comprising: (a) a first
recombinant vector comprising the recombinant polynucleotide of
claim 1, wherein the at least one psRNAV structural protein of the
second portion is selected from a psRNAV capsid and a psRNAV
glycoprotein; and (b) a second recombinant vector comprising the
recombinant polynucleotide of claim 5, wherein the at least one
psRNAV structural protein of the first portion is selected from a
psRNAV capsid and a psRNAV glycoprotein; wherein the psRNAV
structural protein of (a) and the psRNAV structural protein of (b)
are not the same; and wherein the first recombinant vector and the
second recombinant vector are not derived from the same plasmid or
virus vector.
24. A psRNAV replicon packaging system comprising: (a) a first
recombinant vector comprising the recombinant polynucleotide of
claim 9, wherein the replicon-like psRNAV helper RNA sequence of
the second portion is a sequence encoding a psRNAV structural
protein selected from a psRNAV capsid and a psRNAV glycoprotein;
and (b) a second recombinant vector comprising the recombinant
polynucleotide of claim 13, wherein the replicon-like psRNAV helper
RNA sequence of the first portion is a sequence encoding a psRNAV
structural protein selected from a psRNAV capsid and a psRNAV
glycoprotein; wherein the psRNAV structural protein of (a) and the
psRNAV structural protein of (b) are not the same; and wherein the
first recombinant vector and the second recombinant vector are not
derived from the same plasmid or virus vector.
25. An alphavirus replicon packaging system comprising: (a) a first
recombinant vector comprising the recombinant polynucleotide of
claim 3; and (b) a second recombinant vector comprising the
recombinant polynucleotide of claim 7; wherein the alphavirus
structural protein of (a) and the alphavirus structural protein of
(b) are not the same; and wherein the first recombinant vector and
the second recombinant vector are not derived from the same plasmid
or virus vector.
26. An alphavirus replicon packaging system comprising: (a) a first
recombinant vector comprising the recombinant polynucleotide of
claim 11; and (b) a second recombinant vector comprising the
recombinant polynucleotide of claim 15; wherein the alphavirus
structural protein of (a) and the alphavirus structural protein of
(b) are not the same; and wherein the first recombinant vector and
the second recombinant vector are not derived from the same plasmid
or virus vector.
27. A method for obtaining alphavirus replicon particles
comprising: (a) coinfecting a cell with an alphavirus packaging
system of any of claims 19-26; (b) incubating the coinfected cell
under appropriate conditions for replicon particles to be
generated; and (c) obtaining the generated replicon particles from
the cell.
28. An alphavirus replicon particle obtained from the method of
claim 27.
29. A host cell coinfected with the alphavirus replicon packaging
system of any of claims 19-26.
30. An isolated foreign polypeptide produced by the coinfected host
cell of claim 29.
31. An immunogenic composition comprising at least one alphavirus
replicon particle obtained from the method of claim 27 and a
physiologically acceptable carrier or diluent.
32. A method for inducing an immune response in a mammalian or
human host comprising: administering to the host an immunologically
effective amount of the immunogenic composition of claim 31
33. A pharmaceutical formulation comprising at least one alphavirus
replicon particle obtained from the method of claim 27 and a
physiologically acceptable carrier or diluent.
34. A method for producing a prophylactic, therapeutic, or
palliative effect in a mammalian or human host comprising
administering to the host an effective amount of the pharmaceutical
formulation of claim 33.
35. A kit for obtaining alphavirus replicon particles comprising
the packaging system of any of claims 19-26.
36. A kit for obtaining alphavirus replicon particles comprising
the recombinant MVA of claim 18.
37. The recombinant vector of claim 17, wherein the recombinant
vector is a viral vector selected from poxvirus, adenovirus,
herpesvirus, picornavirus, poliovirus, influenza virus, lentivirus,
and retrovirus.
38. A recombinant polynucleotide comprising: a first portion
comprising a first heterologous promoter operatively linked to a
first sequence encoding a psRNAV capsid; and a second portion
comprising a second heterologous promoter operatively linked to a
second sequence encoding a psRNAV glycoprotein.
39. The recombinant polynucleotide of claim 38, wherein the first
and second heterologous promoters are each selected from a poxvirus
promoter, a vaccinia virus synthetic early/late promoter, a T7
promoter, a T3 promoter, and an SP6 promoter; and wherein the
psRNAV capsid of the first portion is an alphavirus capsid; and
wherein the psRNAV glycoprotein of the second portion is an
alphavirus glycoprotein.
40. The recombinant polynucleotide of claim 39, wherein the first
and second heterologous promoters are both vaccinia virus synthetic
early/late promoters; wherein the alphavirus capsid of the first
sequence is a VEE capsid; and wherein the alphavirus glycoprotein
of the second sequence is a VEE glycoprotein.
41. A recombinant MVA comprising the polynucleotide of any of
claims 38-40.
42. A method of amplifying alphavirus replicon particles
comprising: (a) coinfecting a cell with the recombinant MVA of
claim 41 and an alphavirus replicon particle; (b) incubating the
coinfected cell under appropriate conditions for the replicon
particle to be replicated; and (c) obtaining the amplified replicon
particles from the cell.
43. A recombinant polynucleotide comprising: a first portion
comprising a sequence encoding a DNA-dependent RNA polymerase
operatively linked to a first heterologous promoter; and a second
portion comprising a replicon-like psRNAV helper RNA sequence
comprising a reporter gene operatively linked to a second
heterologous promoter.
44. The recombinant polynucleotide of claim 43, wherein the
DNA-dependent RNA polymerase of the first portion is selected from
a T3, T7, or SP6 DNA-dependent RNA polymerase; wherein the first
heterologous promoter is a poxvirus promoter; wherein the reporter
gene of the second portion is selected from a luciferase gene, a
chloramphenicol acetyltransferase (CAT) gene, a beta-galactosidase
gene, a beta-glucoronidase gene, a blue fluorescent protein (BFP)
gene, a yellow fluorescent protein (YFP) gene, and a green
fluorescent protein (GFP) gene; and wherein the second heterologous
promoter binds to a polymerase selected from a T3, T7, or SP6
DNA-dependent RNA polymerase.
45. The recombinant polynucleotide of claim 44, wherein the
DNA-dependent RNA polymerase of the first portion is a T7
DNA-dependent RNA polymerase; wherein the first heterologous
promoter is a vaccinia virus synthetic early/late promoter; wherein
the reporter gene of the second portion is a GFP gene; and wherein
the second heterologous promoter binds to a T7 DNA-dependent RNA
polymerase.
46. The recombinant polynucleotide of claim 43, wherein the psRNAV
helper RNA sequence is selected from an alphavirus helper RNA
sequence, a rubella virus helper RNA sequence, a coronavirus helper
RNA sequence, a dengue virus helper RNA sequence, and a Hepatitis C
virus helper RNA sequence.
47. A recombinant MVA comprising the recombinant polynucleotide of
any of claims 43-46.
48. A method of determining the titer of a solution of psRNAV
replicon particles comprising: (a) coinfecting cells with the
recombinant MVA of claim 47 and a solution of psRNAV replicon
particles; (b) incubating the coinfected cells under appropriate
conditions for expression of a reporter gene; (c) detecting the
expression of the reporter gene; and (d) determining the titer of
the solution of psRNAV replicon particles.
49. A recombinant MVA comprising the polynucleotide of any of
claims 1-16, 38-40, or 43-46, wherein the polynucleotide is
inserted into deletion I, deletion II, deletion III, deletion IV,
deletion V, deletion VI, the sequence encoding hemaglutinin or the
sequence encoding thymidine kinase.
50. The replicon packaging system of any of claims 23-26, wherein
the first recombinant vector and second recombinant vector are
viral vectors selected from poxvirus, vaccinia virus, adenovirus,
herpesvirus, picomavirus, poliovirus, influenza virus, lentivirus,
and retrovirus.
51. The method of claim 27, wherein the cell is selected from a
BHK-21 cell and a FRhL cell.
52. The method of claim 42, wherein the cell is selected from a
BHK-21 cell and a FRhL cell.
53. The method of claim 48, wherein the cells are selected from
BHK-21 cells and FRhL cells.
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to recombinant
polynucleotides, positive-strand RNA virus recombinant expression
vectors, and packaging systems. The packaging systems are based on
the expression of split-helper functions by coinfecting cells with
recombinant vectors, such as recombinant poxvirus vectors.
BACKGROUND OF THE INVENTION
[0002] Recombinant DNA technology has now made it possible to use
viruses to introduce virtually any gene of interest into almost any
cell of interest. Because such viruses are engineered to "express"
the gene of interest, i.e., produce the protein encoded by the
gene, they are called "viral expression vectors".
[0003] Recent attention has focused on alphaviruses, which are
positive-strand RNA viruses (viruses whose nucleic acid is in the
form of RNA, rather than DNA) that are transmitted to mammals via
arthropods (reviewed in (16) and (17)). Positive strand RNA
viruses, and in particular, alphaviruses, are especially attractive
viral expression vectors for several reasons: 1) their genomes are
easily manipulated in cDNA form and are infectious as naked RNA, 2)
their replication cycle is exclusively cytoplasmic, 3) foreign gene
expression is driven by a strong viral promoter, and 4) they have a
broad host-range in vitro.
[0004] Structurally, as set forth in FIG. 1, the alphavirus genome
is a single-stranded RNA, approximately 11.7 kilobases (kb) long,
that is "capped" at the 5' end and "polyadenylated" at the 3' end.
The two-thirds of the genome at the 5' end encodes nonstructural
proteins (nsPs), and the one third at the 3' end encodes structural
proteins (sPs). FIG. 1 also shows that the nonstructural proteins
are responsible for both replication (copying) of the entire RNA
sequence as well as transcription of a "subgenomic" RNA that leads
to translation of the structural proteins (for review see (32) and
(34)).
[0005] For replication, the nsPs are translated directly from the
infecting viral genome, designated (+) RNA, as set forth in step 1
of FIG. 1 (steps are indicated by dark circles that contain
numbers). The translation of the nsPs yields four proteins that
form a "replication/transcription complex", comprising a
"replicase" and a "transcriptase." The replicase/transcriptase
mediates the synthesis of a genome-length complementary strand,
designated (-) RNA, which is also termed the "antigenome", as set
forth in step 2. In step 3, the replicase/transcriptase then
creates an additional copy of (+) full-length RNA using the
antigenome as a template.
[0006] The antigenome also serves as the template for transcription
of the last third of the genome into subgenomic mRNA, as indicated
in the step prior to step 4. As noted above, the 3' one-third of
the genome encodes sPs and, accordingly, the subgenomic mRNA
encodes the sPs. The sPs are encoded in the form of a large
"polyprotein" that is then processed to yield a capsid protein and
two envelope proteins, which are designated E1 and E2. The
transcription of the subgenomic segment is mediated by nucleotides
that span the junction between the nsP coding region and the sP
coding region, and serve as a "promoter". Transcription from the
subgenomic promoter can yield levels of subgenomic mRNA that can
reach 10.sup.6 copies/cell, resulting in 10.sup.8 viral structural
proteins per infected cell (30).
[0007] Once the envelope proteins and capsid proteins are
synthesized, as per step 4, the capsid protein interacts with the
replicated genome RNA to form a "nucleocapsid", which is then
packaged by the envelope proteins. "Packaging signals" that are
located within the nsP coding sequence of the genomic RNA serve to
facilitate this process. Because the subgenomic RNA lacks these
packaging signals, only the genomic RNA is encapsidated.
[0008] Viruses in the Togaviridae and Flaviviridae have similar
enveloped, icosahedral nucleocapsid structures, and are believed to
have evolved from a common ancestral virus (54). Alphaviruses and
rubiviruses (eg. rubella virus), both members of the Togaviridae
family, have similar genomic structures and replication cycles (see
FIG. 22). The replicase/transcriptase complex is translated
directly from the 5'-end of the genome, and the sPs are transcribed
downstream from a subgenomic promoter present on the antisense RNA.
Viruses in the Flaviviridae (eg. Dengue virus, hepatitis C virus,
tick-borne encephalitis virus), all have common genome
organizations and replication strategies. Unlike the togaviruses,
flavivirus genomes serve as the mRNA for a polyprotein that encodes
both the sPs and the nSPs. The expression of these gene products is
regulated at post-translational steps; there is no subgenomic
transcription in the Flaviviridae. Furthermore, the gene
arrangement is inverted, i.e., the sP genes are located upstream of
the nSPs. Although these viruses differ in their genomic
arrangement and replication strategies, they can be substituted for
the viruses described herein. For example, they can be engineered
into replicon expression vectors by removing the sPs coding region
((55), (56)), and packaged into virus-like particles by providing
the sPs in trans (57), using techniques substantially similar to
those described herein.
[0009] Other positive strand RNA viruses that have been engineered
as either live and/or replication-defective expression vectors
include poliovirus (58) and coronavirus (59). Although they also
differ from the alphaviruses, replicon vectors derived from these
viruses may be packaged using techniques similar to those described
herein.
[0010] Understanding the replication/transcription processes of the
alphaviruses, as well as their nucleic acid sequence, has permitted
their use as expression vectors. Several alphaviruses have been
sequenced, and infectious cDNA clones have also been engineered for
Sindbis virus (SV; (31)), Semliki Forest virus (SFV; (20)),
Venezuelan equine encephalitis virus (VEE; (11)), and Ross River
virus (RRV; (18)). Vectors based on SV, SFV and VEE have shown
promise as effective gene expression systems (for reviews see (14),
(21), (15)).
[0011] There are, in general, two types of alphavirus expression
vectors. In one type of vector, the "replication-competent" vector,
a second subgenomic promoter is added to direct the expression of a
foreign (heterologous) gene. This type of double-subgenomic
promoter vector expresses the foreign gene of interest, as well as
all the structural components needed for viral packaging; thus,
these vectors are self-replicating and self-packaging. The apparent
disadvantage of such a system is the production of viable
virus.
[0012] To minimize the potential production of a viable virus, the
alphavirus expression vectors have been further engineered to be
"replication-defective." These vectors are created by removing the
genes that encode for sPs, and substituting one or more foreign
genes under the control of the subgenomic promoter. Since the nsP
coding sequence remains intact, these vectors can form the
replication complex and self-replicate and express the foreign
gene(s). They are not self-packaging, however, because they lack
the sPs which encode the capsid and envelope proteins. To package
these vectors into infectious particles, the vectors can be
complemented "in trans" with "helper" vectors, i.e., vectors that
bear the sPs on a separate RNA molecule. For example, these vectors
may be packaged by cotransfecting the vector with in vitro
transcribed defective-helper (DH) RNAs that encode the viral capsid
and glycoproteins (19), (5) or, alternatively, by transfecting the
replicon RNA into a continuous packaging cell line which expresses
DH RNAs under the regulation of a nuclear promoter (26). With
either system, the helper RNA is either not packaged, or packaged
with very low efficiency, since it lacks the packaging signal
present within the nsP coding region.
[0013] Recombination frequently occurs, however, between alphavirus
replication intermediates (including the replicon and DH RNAs) and
can result in the creation of self-replication and self-packaging
virus (35), (29), (37). This poses potential biosafety and
regulatory concerns about the use of these packaging systems. To
address these concerns, scientists have developed "split-helper"
packaging systems which significantly decrease the probability of
generating vectors that are able to replicate and self-package
(14), (27), (33). The "split-helper" system uses two separate DH
RNAs, one encoding the capsid protein and another encoding the
viral glycoproteins (E2/E1). This is a costly and inefficient
system, however, since the two separate DH RNAs must first be
transcribed in vitro, purified, and subsequently inserted into a
packaging cell that has been prepared for transfection. Numerous
manipulations of the RNA and cells result in inconsistent
production of replicon particles.
[0014] Cells infected with an alphavirus typically produce
10.sup.3-10.sup.4 infectious virus particles/cell. The production
of replicon particles, by contrast, is much less efficient. Cells
transfected with these vectors typically produce an average of 1-50
replicon particles per cell. The low yield of replicon particles is
the result of the cumulative effects of poor in vitro transcription
and cellular transfection. For example, successful expression of
RNA that has been transcribed in vitro requires that the RNA be
capped at the 5' end. For the split-helper systems, which contain
two separate DH RNAs, there are three RNA segments that must be
capped: both helper RNAs and the replicon itself. If the efficiency
of the capping of the replicon in vitro is, for example, 65% and of
each DH RNA is 85%, then the efficiency of the transfection is at
best 42% (0.65.times.0.8.times.0.8). Thus, the efficiency of
expression is limited by the efficiency of the three capping
reactions and the transfection process.
[0015] Compounding the capping problem is the fact that
transfection procedures using chemical reagents are relatively
ineffective. Electroporation methods, where RNAs are introduced
into cells using an electric field rather than chemicals, are more
efficient, but they require numerous manipulations and rigorous
optimizations. Additionally, electroporation methods have not yet
been successfully used in large-scale preparations.
[0016] The ideal packaging system would entail using an efficient
gene delivery system that is optimized for gene expression. Such a
system can be based on plasmids or viral vectors (e.g., poxviruses,
adenoviruses, herpesviruses, poliovirus, influenza viruses,
retroviruses, etc.). Viral vectors can be used to infect a broad
range of cell types in large-scale with great efficiency. Many
viral vectors have been engineered for optimal gene expression and
limited growth in specific cell lines.
[0017] Yet another potential limitation to using these replicon
vectors is the lack of large-scale packaging systems for vector
particles. The preparation of the reagents needed for packaging of,
for example, alphavirus particles is costly, impractical, and not
amenable to meaningful scale-up. Thus, there exists a need in the
art for safe and cost-effective replicon expression vectors and
packaging systems. Such vectors would be used to efficiently
deliver and express psRNAV-derived RNAs for the large-scale
production of infectious replicon particles for the purposes of
subunit vaccine gene delivery, gene therapy, cancer immunotherapy,
and recombinant protein synthesis.
SUMMARY OF THE INVENTION
[0018] The present invention is directed to infectious
positive-strand RNA virus (psRNAV) replicon particles, recombinant
positive-strand RNA virus expression vectors, and packaging systems
for producing psRNAV replicon particles using recombinant poxvirus
vectors. These packaging systems do not require the in vitro
synthesis or transfection of viral RNAs. Instead, the methods and
compositions of the invention use recombinant DNA viruses and/or
plasmids to deliver to a cell the components required to assemble
infectious psRNA replicon particles. Methods and compositions are
provided, including immunogenic compositions and pharmaceutical
formulations for administering to a host. Also provided are
recombinant polynucleotides and vectors for creating poxvirus-based
replicon particle packaging systems. Kits for use with the
disclosed compositions and methods are provided.
[0019] Certain packaging systems of the invention are based on
coinfection with a pair of recombinant poxvirus vectors whose
expression products can package recombinant psRNAV replicon
particles. This system produces infectious replicon particles,
including at least one foreign gene (that is a gene that is taken
out of its natural state), which may be a viral gene. In an
exemplary embodiment, the pair of poxvirus vectors is based on a
vaccinia virus, specifically, a severely host-restricted,
attenuated strain of vaccinia virus (modified vaccinia virus
Ankara, or MVA). In this embodiment, the psRNAV is an alphavirus,
Venezuelan equine encephalitis virus (VEE). The pair of MVA vectors
produce the VEE capsid and envelope proteins that thereafter
package the VEE replicon RNA to produce an infectious VEE replicon
particle (VRP).
[0020] The MVA-based VRP packaging systems rely on the production
of replicon RNAs and helper proteins. The expression of the helper
proteins is either inducible or constitutive based on the form of
the RNAs transcribed by the MVA vector. Inducible helper RNAs are
replicated and transcribed by the VEE replicase/transcriptase prior
to being translated. Constitutive helper RNAs are transcribed as
mRNAs and therefore are translated directly.
[0021] The replicon particle packaging systems of the invention
yield greater titers of replicon particles than the conventional
split-helper RNA transfection method discussed above. Using the
packaging systems disclosed herein, helper function RNAs can be
expressed either as DH RNAs, or as mRNAs, depending on the
packaging system employed. The probability of generating
self-replicating infectious virus during the replicon packaging
process is further reduced in the constitutive system since all of
the psRNAV regulatory sequences are absent from the helper RNAs.
This system provides the greatest level of safety of all known
packaging systems.
[0022] Additionally, the invention provides methods for using other
poxvirus vectors in the large-scale production of recombinant gene
products (2). These methods are amenable to adaptation for the
mass-production of replicon particles.
[0023] The novel recombinant polynucleotides and recombinant
vectors, including recombinant viruses, can be prepared using
standard cloning and molecular biology techniques that are well
known in the art. Descriptions of such techniques can be found,
among other places, in Sambrook et al., Molecular Cloning (1989)
(38) and Ausbel et al., Current Protocols in Molecular Biology
(1993, including supplements (39)).
[0024] The invention is based upon recombinant polynucleotides that
serve as components for recombinant vectors, including recombinant
viruses, that in turn are components of replicon particle packaging
systems. In certain embodiments, these recombinant viruses and
packaging systems are used in methods of the invention to obtain
replicon particles.
[0025] In certain embodiments, recombinant polynucleotides within
the invention comprise at least a first portion and a second
portion. The first portion includes a sequence with at least a
first heterologous promoter that is operatively linked to a
sequence that encodes a DNA-dependent RNA polymerase. By
"operatively linked" is meant a linkage that permits regulatory
control, such as a promoter that controls expression. The second
portion includes a second heterologous promoter that is operatively
linked to a sequence that encodes at least one psRNAV structural
protein, but it does not encode all of the psRNAV structural
proteins. Thus, the second portion may encode a psRNAV capsid or it
may encode a psRNAV glycoprotein, but not both. The terms first
portion and second portion are not intended to indicate any
sequential position within the recombinant polynucleotides. Thus
the first portion may be either upstream or downstream of the
second portion.
[0026] Further, the terms first portion and second portion are not
intended to be limiting. For example, in certain embodiments the
recombinant polynucleotides comprise three or more portions. For
instance, a second portion may comprise a sequence that encodes an
E1 glycoprotein and a third portion may comprise a sequence that
encodes an E2 glycoprotein.
[0027] The promoters of the first and second portions may be the
same or different. In the first portion, the DNA-dependent RNA
polymerase to which the first heterologous promoter is attached may
be a viral or bacteriophage polymerase, for example, without
limitation, a bacteriophage T3, T7, or SP6 DNA-dependent RNA
polymerase.
[0028] In certain embodiments, the first and second heterologous
promoters are different. For example, without limitation, the first
promoter may comprise a poxvirus promoter and the second promoter
comprise a bacteriophage promoter. In one exemplary embodiment, the
recombinant polynucleotide has a vaccinia virus synthetic
early/late promoter operatively linked to a sequence encoding
bacteriophage T7 DNA-dependent RNA polymerase, and a second
heterologous promoter, that binds to T7 DNA-dependent RNA
polymerase, operatively linked to a sequence encoding at least one
psRNAV structural protein, such as a Venezuelan equine encephalitis
virus glycoprotein.
[0029] In certain embodiments of the invention, recombinant
polynucleotides comprise a first portion which includes a first
heterologous promoter operatively linked to a sequence encoding at
least one, but not all, of the psRNAV structural proteins. Thus,
the first portion will contain a sequence encoding either the
psRNAV capsid protein or a psRNAV glycoprotein, but not both. The
second portion includes a second heterologous promoter operatively
linked to a psRNAV "replicon" which is capable of replication. The
psRNAV replicon of the second portion includes a psRNAV subgenomic
promoter operatively linked to a sequence encoding at least one
foreign polypeptide. In certain embodiments, the first and second
heterologous promoters may bind the same polymerase, for example,
but not limited to, T7 DNA-dependent RNA polymerase, or they may
bind different polymerases, for example, but without limitation,
DNA-dependent RNA polymerases from poxvirus or bacteriophage T3,
T7, or SP6. In certain embodiments, the psRNAV is an
alphavirus.
[0030] In one exemplary embodiment, the recombinant polynucleotide
has a bacteriophage T7 promoter operatively linked to a sequence
encoding an alphavirus capsid protein, and a second T7 promoter
operatively linked to a sequence encoding an alphavirus replicon.
In certain embodiments, the alphavirus capsid and alphavirus
replicon are from Venezuelan equine encephalitis virus.
[0031] In yet other embodiments, the recombinant polynucleotide has
a first portion which includes a first heterologous promoter
operatively linked to a sequence encoding a DNA-dependent RNA
polymerase. The second portion includes a sequence encoding a
replicon-like psRNAV helper RNA operatively linked to a second
heterologous promoter. Thus, when a DNA-dependent RNA polymerase
binds to the second heterologous promoter, a replicon-like psRNAV
helper RNA is transcribed. When the replicon-like helper RNA is
exposed to the psRNAV replication complex, it is replicated by the
replicase to produce an "antigenomic" strand that is transcribed by
the transcriptase to produce a subgenomic transcript. The
subgenomic transcript is then translated to produce either a psRNAV
capsid protein or a psRNAV glycoprotein, but not both.
[0032] In these embodiments, the sequence encoding a DNA-dependent
RNA polymerase encodes a bacteriophage polymerase, such as from
bacteriophage T3, T7, SP6, or the like, and the replicon-like
helper sequence comprises the sequence encoding a psRNAV capsid In
these embodiments, the first heterologous promoter comprises, for
example, a poxvirus synthetic early/late promoter, and the second
heterologous promoter binds to a bacteriophage DNA-dependent RNA
polymerase, such as T3, T7, or SP6 polymerase.
[0033] In other embodiments, recombinant polynucleotide has a
vaccinia virus synthetic early/late promoter operatively linked to
a sequence encoding a bacteriophage T7 polymerase; and a second
heterologous promoter, that binds to a T7 DNA-dependent RNA
polymerase, is operatively linked to a replicon-like helper
sequence comprising a sequence encoding a psRNAV capsid.
[0034] In yet other embodiments, a recombinant polynucleotide of
the invention has a first portion including a sequence encoding a
replicon-like psRNAV helper RNA operatively linked to a first
heterologous promoter; and a second portion comprising a second
heterologous promoter operatively linked to a psRNAV replicon. The
psRNAV replicon includes a psRNAV subgenomic promoter operatively
linked to a sequence encoding at least one foreign polypeptide. In
these embodiments, the first and second heterologous promoters may
be the same or they may be different. In certain embodiments, the
replicon-like helper sequence comprises a sequence encoding a
psRNAV glycoprotein. In certain embodiments, the first and second
heterologous promoters binds to a bacteriophage DNA-dependent RNA
polymerase, for example, without limitation, T3, T7, or SP6
polymerase. In certain embodiments, the psRNAV is an
alphavirus.
[0035] In one exemplary embodiment, the first and second promoters
of the recombinant polynucleotide bind to a T7 DNA-dependent RNA
polymerase, and the replicon-like helper sequence comprises a
sequence encoding an alphavirus glycoprotein, for example, but not
limited to, Venezuelan equine encephalitis virus glycoprotein.
[0036] Once the recombinant polynucleotides discussed above have
been generated, they can be used to create recombinant vectors,
such as but not limited to, a cloning vector or expression vector.
Exemplary cloning vectors or expression vectors include bacterial
plasmids, phagemids, recombinant viruses, yeast vectors, and the
like. Favorable attributes of cloning vectors may include ease of
cloning and in vitro manipulations. Favorable attributes of
expression vectors may include robust gene expression, broad
host-range of infectivity, limited growth or no growth in
restrictive cell lines, infectious to mammalian host cells, control
of host-cell antiviral responses, nonpathogenic to laboratory
personnel. Preferred recombinant plasmid or virus vectors include
poxviruses, adenoviruses, herpesviruses, poliovirus, influenza
viruses, and retroviruses. A particularly preferred recombinant
poxvirus vector is modified vaccinia virus Ankara (MVA).
[0037] In certain embodiments, for biosafety reasons, the packaging
systems of the invention may comprise three recombinant vectors,
each encoding a different psRNAV structural polypeptide. In certain
embodiments, the three recombinant vectors include, but are not
limited to, a first recombinant vector comprising a sequence
encoding a capsid, a second recombinant vector comprising a
sequence encoding an E1 glycoprotein, and a third recombinant
vector comprising a sequence encoding an E2 glycoprotein.
[0038] The skilled artisan will understand that the recombinant
virus vectors used in the packaging systems of the invention may,
but need not, be derived from the same recombinant virus. For
example, with certain virus systems, infection by a first virus may
cause the infected cell to become refractory to superinfection by
another virus from the same virus system. Thus, in certain
embodiments, it may be preferred to employ a packaging system
wherein the first recombinant virus is derived from a different
virus system than the second recombinant virus. For example, but
not limited to, a packaging system comprising a poxvirus-derived
vector and an adenovirus-derived vector.
[0039] Additionally, double-promoter insertion/expression vectors
can be used. For example, the synthetic early/late promoter of
vaccinia virus has been engineered in a back-to-back configuration
so that two genes can be inserted simultaneously into the same site
of the poxvirus (50). This same type of vector could also be used
to insert multiple expression cassettes in the MVA packaging
systems.
[0040] In certain embodiments, recombinant MVAs comprise one or
more recombinant polynucleotides of the invention inserted into MVA
deletions II or III (See FIG. 2). In other embodiments,
recombinants comprise one or more recombinant polynucleotides of
the invention inserted into the MVA hemagglutinin gene (42), the
thymidine kinase gene (52), or into other nonessential regions of
the MVA genome. Insertions into other MVA deletion sites are also
within the scope of the invention. For example, six MVA deletions
have been mapped, including deletion I, a 2.9 kilobasepair (kbp)
deletion within the HindIII C restriction fragment; deletion II, a
5.0 kbp deletion within the HindIII N restriction fragment;
deletion III, a 3.5 kbp deletion within the HindIII A restriction
fragment; deletion IV, a 10.2 kbp deletion within the HindIII B
restriction fragment; deletion V, a 4.7 kbp deletion within the
HindIII C fragment; and deletion VI, a 3.8 kbp deletion within the
HindIII A restriction fragment (See FIG. 2 for HindIII restriction
map). Descriptions of the mapping of MVA deletions can be found,
among other places, in references (41), (49) and (51).
[0041] Replicon packaging systems are also provided. The novel
packaging systems disclosed herein comprise at least two
recombinant vectors of the invention. The packaging systems of the
invention may be inducible or constitutive. An exemplary psRNAV
replicon packaging system comprises two recombinant MVAs, each of
which has a first and second portion. A first exemplary recombinant
MVA has a first portion that comprises a vaccinia virus synthetic
early/late promoter operatively linked to a sequence encoding
bacteriophage T7 DNA-dependent RNA polymerase, and a second portion
that comprises a second heterologous promoter that binds to T7
DNA-dependent RNA polymerase, operatively linked to a sequence
encoding at least one alphavirus structural protein inserted into
deletion III, wherein that sequence encodes VEE glycoprotein (see
FIG. 2). A second exemplary recombinant MVA has a first portion
comprising a bacteriophage T7 promoter operatively linked to a
sequence encoding a Venezuelan equine encephalitis virus capsid
protein inserted into deletion III, and a second portion comprising
a T7 promoter operatively linked to a sequence encoding a VEE
replicon, inserted into deletion II (see FIG. 2).
[0042] Methods for producing infectious replicon particles are also
provided. According to certain embodiments, cells are coinfected
with recombinant viruses comprising a novel replicon particle
packaging system of the invention. In one exemplary embodiment,
cells are coinfected with one or more MVA recombinant viruses
comprising a novel replicon particle packaging system of the
invention. In another exemplary embodiment, methods of amplifying
infectious replicon particles are provided. Cells are coinfected
with psRNAV replicon particles and MVA recombinant(s) that express
psRNAV capsid and glycoproteins. In both exemplary embodiments, the
coinfected cells are incubated under appropriate conditions for
replicon particles to be generated. Once generated, the replicon
particles are obtained.
[0043] Methods of titering replicon particles are provided.
According to certain embodiments, cells are infected with an MVA
recombinant that delivers to the cell a suitable reporter gene.
When a cell is coinfected with a replicon particle, the reporter
gene is activated, and the cell can then be detected. Examples of
reporter genes that may be used with this system include green
fluorescent protein, blue fluorescent protein, yellow fluorescent
protein, beta-galactosidase, beta-glucoronidase choramphenicol
acetyl-transferase, and luciferase.
[0044] The replicon particles of the invention may be used in
immunogenic compositions or pharmaceutical formulations to be
administered to a host. Such compositions and formulations may
further comprise appropriate physiologically acceptable carriers,
adjuvants, diluents, excipients, immunostimulatory compounds, and
the like. These compositions and formulations may be administered
using any effective method. Exemplary administration methods
include, intravenous, intramuscular, or intradermal injection,
intranasal instillation, orally, topical application to a dermal or
mucosal surface, and the like.
[0045] In certain embodiments, methods for inducing an immune
response in a mammalian or human host are provided. Such methods
comprise administering to the host an immunologically effective
amount of an immunogenic composition of the invention to induce an
immune response in the host. In certain embodiments, methods for
producing a prophylactic, therapeutic, or palliative effect in a
mammalian or human host are provided. These methods comprise
administering to the host an effective amount of a pharmaceutical
formulation of the invention to produce a prophylactic,
therapeutic, or palliative effect.
[0046] Also within the scope of the invention are foreign proteins
that are expressed by the recombinant polypeptides; recombinant
vectors, including recombinant viruses; replicon particle packaging
systems of the invention; and replicon particles obtained using the
methods of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1. Schematic diagram showing the steps of the
alphavirus replication cycle. Within the infected cell, the 5'
two-thirds of the alphavirus genome is translated, from a single
translation initiation site, to generate the four alphavirus
nonstructural proteins (nsPs) (step 1). The nsPs are required for
the synthesis of the complementary (-) RNA strand (step 2) that
serves as the template for replicating the genomic RNA and
transcription of the subgenomic RNA (step 3). The subgenomic RNA is
translated to produce the structural proteins (sPs) (step 4) that
interact with the packaging signals on the genomic RNA, but not the
subgenomic RNA, to assemble infectious alphavirus particles (step
5). The horizontal arrow shown on the complementary (-) RNA strand
indicates the alphavirus subgenomic promoter. (Figure from
Schiessinger, S. 1999. ASM News 65:688-95.)
[0048] FIG. 2. Schematic representation of the MVGKT7 expression
vector. Shown is the HindIII restriction map of the virus. The
bacteriophage T7 gene-1 is located in the HindIII J fragment. Also
shown are MVA deletions II and III that are useful for the
insertion of recombinant polynucleotides and foreign genes. Other
deletions (such as I, IV, V, and VI) have been mapped relative to
these HindIII restriction sites and may also be useful sites for
foreign gene insertion.
[0049] FIG. 3. Schematic representation of an exemplary alphavirus
replicon particle constitutive packaging system. The HindIII
restriction map is shown superimposed on CMVA1 and CMVA2.
Abbreviations used in FIGS. 3 and 4--P.sub.T7: bacteriophage T7
promoter; P.sub.7.5: vaccinia 7.5K promoter; P.sub.S-E/L: vaccinia
synthetic early/late promoter; P.sub.11K: vaccinia 11K promoter;
P.sub.G8R: G8R promoter; capORF: capsid open reading frame; gP ORF:
glycoprotein open reading frame; DH gP: sequence encoding
glycoprotein defective helper RNA; DHcap: sequence encoding capsid
defective helper RNA.
[0050] FIG. 4. Schematic representation of an exemplary alphavirus
replicon particle inducible packaging system. Abbreviations are as
identified in the brief description of FIG. 3, above.
[0051] FIG. 5. Schematic map illustrating the generation of plasmid
pGK16.2. Stippled arrowheads represent the vaccinia synthetic
early/late promoter (PE/L) and the vaccinia 7.5K promoter (P7.5K).
TK-L and TK-R indicate regions of homology with the vaccinia virus
thymidine kinase (TK) locus.
[0052] FIG. 6. Schematic map illustrating the generation of
plasmids pDF17, pDF30, and pDF33. Stippled arrowheads represent the
vaccinia synthetic early/late promoter (PE/L), the vaccinia 7.5K
promoter (P7.5K), and the vaccinia 11K promoter (P11 K). MF-1 and
MF-2, in this figure, indicate regions of homology with the region
designated "deletion III" of MVA.
[0053] FIG. 7. Schematic map illustrating the generation of
plasmids pGK53 and pGK51. Stippled arrowheads represent the
vaccinia synthetic early/late promoter (PE/L), the vaccinia 7.5K
promoter (P7.5K), the vaccinia 11K promoter (P11K), the
bacteriophage T7 promoter (P-T7), and the alphavirus subgenomic
promoter (P-26S). .DELTA.nsP is a large deletion within the nsP
expression cassette.
[0054] FIG. 8. Schematic representation of the genomes of
recombinant MVAs used in exemplary poxvirus-based alphavirus
replicon particle packaging systems. (A) Inducible system. (B)
Constitutive system. Designations of recombinant MVAs are shown at
right. Specific promoters used are indicated as stippled
arrowheads. VEE replication sequences are indicated as black boxes.
The alphavirus subgenomic promoters are indicated as curved black
arrows. The VEE nonstructural protein genes are denoted as
replicase. GFP, green fluorescent protein; gus,
.beta.-glucuronidase gene; T7 gene-1, T7 RNA polymerase gene; lacZ,
.beta.-galactosidase gene; gP, E3/E2/6K/E1 glycoprotein; cap,
capsid; DH, defective-helper; ORF, open reading frame. Not drawn to
scale.
[0055] FIG. 9. Schematic map illustrating the generation of
plasmids pDF13, pDF49, pDF51, and pGK61. Stippled arrowheads
represent the H5R promoter (P-H5R) and the G8R promoter (P-G8R). In
this figure, MF-1 and MF-2 indicate regions of homology with the
region designated "deletion II" of MVA.
[0056] FIG. 10. Schematic map illustrating the generation of
plasmids pVR3 and pVRGFP. Stippled arrowheads represent the
bacteriophage T7 promoter (P-T7) and the alphavirus subgenomic
promoter (P-26S).
[0057] FIG. 11. Schematic map illustrating the generation of
plasmid pGK63. Stippled arrowheads represent the bacteriophage T7
promoter (P-T7), the G8R promoter (P-G8R), and the alphavirus
subgenomic promoter (P-26S).
[0058] FIG. 12. Schematic map illustrating the generation of
plasmids pGK64 and pGK65. Stippled arrowheads represent
bacteriophage T7 promoter (P-T7), the vaccinia synthetic early/late
(P-E/L) and 11K (P11K) promoters, and the alphavirus subgenomic
promoter (P-26S).
[0059] FIG. 13. VEE inhibits MVA late gene expression. BHK-21 cells
were infected at a multiplicity of infection (MOI) equivalent to 10
plaque forming units (PFU) of recombinant MVAs alone, or 10
infectious units (IU) of VRPIGFP, or both, and harvested at 24
hours post infection (hpi). Cytosine beta-D arabinosidase, or AraC
(44 .mu.g/ml), was added to cells where indicated. Lysates were
prepared and assayed for .beta.-galactosidase activity
(OD.sub.490).
[0060] FIG. 14. VRP packaging with an exemplary MVA-based inducible
VRP-packaging system. Approximately 1.times.10.sup.6 Baby hamster
kidney (BHK)-21 cells were co-infected with MVA/VEEGFP/DHgP (IMVA2)
and MVGKT7/DHcap (IMVA1) at the indicated MOI, or alternatively
they were co-transfected with replicon-GFP RNA, capsid and
glycoprotein DH RNAs. VRP/GFP titers in the infected- and
transfected-cell media were determined and plotted as total IU of
VRP/GFP per 35 mm dish. Numbers above bars indicate the average
number of VRP/GFP produced per cell.
[0061] FIG. 15. VRP packaging with an exemplary MVA-based
constitutive VRP-packaging system. BHK-21 cells were co-infected
with MVA/VEEGFP/cap and MVGKT7/gP at the indicated MOI, or
co-transfected as indicated in FIG. 5. Media from infected- and
transfected-cells were titered for VRP/GFP. Titers are plotted as
total IU of VRP/GFP per 1.times.10.sup.6 cells. Numbers above bars
indicate the average number of VRP/GFP yield per cell.
[0062] FIG. 16. Illustrates the expression of structural proteins
by exemplary MVA-based VRP-packaging systems. Approximately,
1.times.10.sup.6 BHK-21 cells were infected with either MVGKT7/gP
and/or MVANEEGFP/cap (constitutive system) or MVANEEGFP/DHgP and/or
MVGKT7/DHcap (inducible system) at a MOI equivalent to 5 PFU of
each virus/cell. Alternatively, BHK-21 cells were co-electroporated
with replicon-GFP RNA, and the capsid and glycoprotein DH RNAs. (A)
Cell lysates were prepared at 24 h and analyzed by immunoblotting
using a VEE-specific antiserum. Molecular weight markers are
indicated at left. The expected immuno-reactive protein bands of
capsid, E2/E1 are indicated at right. The size of a molecular
weight marker is shown at the extreme left. MWM, molecular weight
markers; Cap, MVA/VEEGFP/cap-infected; gP, MVGKT7/gP-infected; RNA,
split-helper RNA electroporated; DHCap, MVGKT7/DHCap-infected;
DHgP, MVA/VEEGFP/DHgP-infected. (B) Infected- and transfected-cell
media were harvested and titered on naive BHK-21 cells. Total IU of
VRP/GFP per 35 mm dish were determined. Numbers above bars indicate
the average number of VRPs produced per cell.
[0063] FIG. 17. VRP packaging on cell lines that are restrictive
for MVA growth. Approximately 2.5.times.10.sup.6 of the indicated
cell lines were co-infected with MVA/VEEGFP/cap and MVGKT7/gP at 10
PFU of each virus per cell. Infected-cell media were titered for
VRP/GFP at 24 hpi and plotted as total IU produced per T-25 flask.
Numbers above bars indicate the average number of VRPs produced per
cell.
[0064] FIG. 18. Depicts the nucleotide sequence of vector pLW17
(obtained from B. Moss), see Example 1B. (SEQ ID NO: 3)
[0065] FIG. 19. Shows a schematic representation of the plasmid
transfer vector, p2104, used for construction of an MVA
recombinant, which will be used to deliver a reporter gene to
cells.
[0066] FIG. 20. Shows a schematic representation of the MVA
recombinant, IMVA3, after recombination of the p2104 transfer
vector into deletion III.
[0067] FIG. 21. Depicts the universal replicon titration system
diagrammatically.
[0068] FIG. 22. Depicts the genomes of two members of the
Alphaviridae (alphavirus and rubivirus) and one representative
virus of the Flaviviridae. Nonstructural genes are indicated in
shaded boxes, and structural genes are shown as stippled boxes.
[0069] FIG. 23. Titering of VRPgD using the IMVA3-based GFP
indicator system. Confluent cultures of VERO cells were infected
with IMVA3 indicator virus (panels A and C), and/or VRPgD replicon
particles (panels B and C). Cells were observed with UV
fluorescence microscopy at 24 hours post infection.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0070] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All references cited in this application,
including articles, books, patents, and patent applications, are
expressly incorporated by reference for any purpose.
[0071] Definitions
[0072] As used herein, the term "positive-strand RNA virus" or
"psRNAV" refers to RNA viruses that exist as a positive-strand RNA.
Positive-strand RNA viruses include, but are not limited to,
alphaviruses, including but not limited to Ross River virus,
Semliki forest virus, Sindbis virus, and Venezuelan equine
encephalitis virus; flaviviruses, including but not limited to
Dengue virus; hepaciviruses, including but not limited to Hepatitus
C virus; coronaviridae, including but not limited to coronavirus;
and rubiviruses, including but not limited to rubella virus.
[0073] The term "glycoprotein" encompasses the alphavirus
glycoproteins, as well as the functionally homologous proteins of
other positive-strand RNA viruses. The term "glycoprotein" when
used in reference to alphavirus proteins or genes, are both used in
a broad sense, encompassing the alphavirus E1 glycoprotein, the
alphavirus E2 glycoprotein, the alphavirus E2/E1 glycoprotein
precursor, and/or an alphavirus polyprotein comprising E3/E2/6K/E1,
or combinations thereof.
[0074] The term "replicon" refers to a replication-defective psRNAV
that has at least one foreign gene inserted into the psRNAV genome
in place of the sequence that encodes the psRNAV structural
proteins. The at least one foreign gene is operatively linked to,
and is thus transcriptionally regulated by, the psRNAV subgenomic
promoter. In certain embodiments, the inserted foreign gene
replaces only some of the sequence encoding the psRNAV structural
proteins. Thus, the replicon encodes both a foreign gene and some,
but not all, of the psRNAV structural proteins.
[0075] The term "defective helper RNA," also referred to as "DH
RNA," describes an RNA that has been designed to contain cis-acting
sequences essential for replication, and a subgenomic promoter for
transcription of one or more structural protein genes. Expression
of the structural proteins is achieved by providing the psRNAV
replicase/transcriptase in trans.
[0076] The term "foreign gene" when used herein refers to a nucleic
acid sequence that has been removed form its natural genetic
environment and placed into a different genetic environment. For
example, but not limited to, a gene encoding a human amyloid
peptide that is operatively linked to an alphavirus subgenomic
promoter, or a Sindbis virus glycoprotein gene operatively linked
to a subgenomic promoter from Venezuelan equine encephalitis. The
term "foreign polypeptide," as used herein refers to a polypeptide
that is encoded by a foreign gene.
[0077] As used herein, the term "immunogenic composition" refers to
one or more substances that stimulate or enhance a humoral or
cellular immune response. Examples of such immunogenic compositions
include, but are not limited to, antigens, T-cell epitopes,
peptides or nucleotides that stimulate or enhance an immune
response.
[0078] The term "operatively linked" refers to a promoter and
coding sequence combination wherein the promoter transcriptionally
regulates the expression of the coding sequence. The term
"heterologous promoter", as used herein, refers to a promoter that
is operatively linked to a different coding sequence than the
promoter is naturally associated with. Exemplary heterologous
promoters may be prokaryotic, eukaryotic, or viral, and include,
but are not limited to, poxvirus promoters, including vaccinia
virus synthetic early/late promoters, bacteriophage T7, T3, and SP6
promoters, cytomegalovirus (CMV) promoters, Rous sarcoma virus
(RSV) promoters, MMTV promoters, Murine leukemia virus promoters,
mammalian pol I promoters, mammalian pol II promoters, and
mammalian pol III promoters. Exemplary heterologous promoters also
include inducible promoters, such as estrogen-responsive promoters,
tetracycline-responsive promoters, metallothionein promoters,
calcium-responsive promoters, and lac promoters. In certain
embodiments, an operatively linked heterologous promoter is a
vaccinia virus promoter operatively linked to a bacteriophage
coding sequence. Certain embodiments provide a bacteriophage
promoter operatively linked to an alphavirus coding sequence.
[0079] The term "pharmaceutical formulation", as used herein,
refers to one or more substances that, when provided to a host in
an effective amount, produces a prophylactic, therapeutic, or
palliative effect in the host. Pharmaceutical formulations may or
may not stimulate an immune response in the host. Examples of such
pharmaceutical formulations include, without limitation, alphavirus
replicon particles encoding insulin, growth hormone, monokines,
cytokines, virokines, or other genes that are desirable for gene
therapy.
[0080] The term "polymerase" refers to an enzyme that can
synthesize nucleic acid polymers from nucleotides in a
template-dependent manner. The nucleic acid polymer that is
produced is complementary to the template. Polymerases that
synthesize RNA polymers are referred to as RNA polymerase while
polymerases that synthesize DNA polymers are referred to as DNA
polymerase. Additionally, polymerases are categorized based on
whether they function using a DNA template or a RNA template. Thus,
for example, a DNA-dependent RNA polymerase synthesizes a
complementary RNA copy using a DNA template.
[0081] The term "polypeptide", as used herein, refers to two or
more amino acids linked together by at least one peptide bond. The
term is used in a general sense to include peptides, oligopeptides,
and proteins.
[0082] The term "replicon-like helper RNA" refers to a sequence
that is transcribed to produce a helper RNA template upon which a
psRNAV replication complex can act. One element in the psRNAV
replication complex, the psRNAV replicase, uses the helper RNA
template to synthesize a single-stranded, negative-sense
"antigenome". That antigenome serves serves as a template for
another element of the replication complex, the psRNAV
transcriptase. The transcriptase synthesizes a mRNA that, when
translated, produces either a psRNAV capsid protein or a psRNAV
glycoprotein, but not both.
[0083] The term "synthetic early/late promoter" refers to a
nucleotide sequence that is useful as a transcriptional promoter
and that has been genetically optimized for expression based on
detailed mutagenesis of a family of promoters (e.g. vaccinia virus
early or late promoters).
[0084] The term "transcription" refers to the process wherein a RNA
polymerase synthesizes a complementary RNA copy of a DNA or RNA
template.
[0085] The term "translation" refers to the process wherein
proteins are synthesized by the translation complexes based on a
RNA template, generally, mRNA.
EXEMPLARY EMBODIMENTS OF THE INVENTION
[0086] The packaging systems of the invention are based on
coinfection with two recombinant vectors bearing a psRNAV replicon
and helper RNAs or genes. These systems do not require transfection
of in vitro synthesized RNA molecules. To more clearly illustrate
the invention, exemplary recombinant vector-based alphavirus
replicon particle packaging systems were generated using a
bacteriophage T7 DNA-dependent RNA polymerase, a modified vaccinia
virus (MVA) vector, and VEE. Both inducible and constitutive
alphavirus replicon particle packaging systems are provided.
[0087] One difference between the two types of systems is in the
structure of the helper function RNAs. In some exemplary inducible
type systems, defective helper (DH) RNAs are transcribed by a T7
DNA-dependent RNA polymerase and subsequently expressed when
replicated and transcribed in the presence of an inducer, VEE
replicase. In some exemplary constitutive type systems, the helper
functions are transcribed as mRNAs by the vaccinia virus
DNA-dependent RNA polymerase, and are expressed throughout the
course of infection, even in the absence of the VEE replicase. The
constitutive packaging system can also be engineered to express the
structural protein genes under the regulation of bacteriophage
promoters.
[0088] Replicon particles are generated when a cell is coinfected
with both of the recombinant vectors of the packaging system. The
replicon particles that are generated are capable of initiating a
single round of infection. However, the replicon particles cannot
form viable virus because only replicon genomes containing the
foreign gene(s) are packaged. This results from the creation of
packaging systems that express replicons that contain packaging
signals and helper RNAs that lack packaging signals.
[0089] Since the psRNAV replicon is not on the same recombinant
viral vector as the T7 DNA-dependent RNA polymerase gene, the
replicon is not expressed unless the second recombinant viral
vector, containing the polymerase gene, is present in the cell.
Further, only the replicon, not the helper RNAs, contain a
packaging signal. Consequently, only the expressed replicon is
packaged to produce infectious particles.
[0090] The skilled artisan will understand that DNA-dependent RNA
polymerases obtained from sources other than bacteriophage T7 are
within the scope of the invention, as are poxviruses and
positive-strand RNA viruses, other than MVA and VEE. Further, in
addition to the consensus nucleotide or amino acid sequences for a
given polymerase, poxvirus, or psRNAV, sequences from variants, for
example, additional clinical isolates or in vitro generated variant
viruses, are contemplated in the invention. Additionally, due to
the degeneracy of the nucleotide code, many different nucleotide
sequences will encode the same amino acid sequence. Thus degenerate
nucleotide sequences are within the intended scope of the
invention.
[0091] In more detail, the constitutive packaging systems comprise
two recombinant poxvirus vectors, which, for illustration purposes,
are constitutive MVA vector 1 (CMVA1, also referred to as
MVGKT7/gp) and constitutive MVA vector 2 (CMVA2, also referred to
as MVA/VEEGFP/cap) (See FIG. 3). CMVA1 comprises the bacteriophage
T7 gene-1, encoding a DNA-dependent RNA polymerase, operatively
linked to a vaccinia virus synthetic early/late promoter, inserted
into the thymidine kinase locus. CMVA1 further comprises the VEE
E2/E1 open reading frame operatively linked to a vaccinia virus
synthetic early/late promoter inserted into MVA deletion III (see
FIG. 3). CMVA2 comprises the VEE capsid open reading frame
operatively linked to a vaccinia virus synthetic early/late
promoter inserted into MVA deletion II. CMVA2 further comprises an
alphavirus replicon, containing the nsPs and at least one foreign
gene, operatively linked to a T7 promoter inserted into MVA
deletion II (see FIG. 3). Cells infected with both CMVA1 and CMVA2
produce T7 polymerase, which transcribes the full-length replicon.
The capsid and E2/E1 mRNAs are translated directly from mRNAs to
produce the alphavirus structural proteins which encapsidate the
replicon, thus producing alphavirus replicon particles. Of note,
the helper functions are transcribed in the constitutive packaging
systems, even if the alphavirus replicase is absent.
[0092] An illustrative inducible packaging systems also comprise
two recombinant poxvirus vectors: inducible MVA vector 1 (IMVA1,
also referred to as MVGKT7/DHcap) and inducible MVA vector 2
(IMVA2, also referred to as MVANEEGFP/DHgP) (See FIG. 4). Like
CMVA1, IMVA1 comprises the bacteriophage T7 gene-1, encoding a
DNA-dependent RNA polymerase, operatively linked to a vaccinia
virus synthetic early/late promoter, inserted into the thymidine
kinase locus. In contrast to CMVA1, IMVA1 further comprises a
replicon-like capsid helper RNA operatively linked to a T7 promoter
inserted into MVA deletion III. IMVA2 comprises a replicon-like
E2/E1 helper RNA operatively linked to a T7 promoter, inserted into
MVA deletion III (See FIG. 4). IMVA2 further comprises a VEE
replicon operatively linked to a T7 promoter inserted into deletion
II of MVA (See FIG. 4). All of the replicon-like helper RNAs of the
inducible packaging systems must be replicated by the alphavirus
replicase before they are translated into helper proteins. Thus,
cells infected with IMVA1 and IMVA2 will produce alphavirus
replicon particles only if an alphavirus replicase is present.
[0093] Once psRNAV replicon particles have been produced, they may
be amplified by coinfecting cells with the replicon particles and
an MVA recombinant expressing psRNAV capsid and glycoproteins. In
this system, the psRNAV replicon is able to replicate, but it lacks
the structural genes required to form new replicon particles. Upon
coinfection with an MVA recombinant expressing the structural
proteins, replicon particles bearing the psRNAV replicon are
assembled. In certain embodiments, the MVA recombinant expresses
each structural protein under the control of a heterologous
promoter. In certain embodiments, the promoter is a vaccinia virus
synthetic early/late promoter, or another suitable poxvirus
promoter(s), bacteriophage T7, T3, or SP6 promoters, or another
promoter that is can direct transcription in the cytoplasm of the
cell. In certain embodiments, vectors expressing bacteriophage T7,
T3, or SP6 are provided in trans, and/or are stably expressed in
the host cell. The skilled artisan will quickly recognize which
promoters would be suitable in the amplification system described
herein.
[0094] The skilled artisan will understand that, following the
disclosed examples and applying only ordinary skill, alternate
polymerases, poxvirus vectors, positive-strand RNA viruses, psRNAV
structural or nonstructural protein sequences, replicons, and
replicon-like helper RNAs, may be used interchangeably in the
recombinant vectors and replicon packaging systems of the
invention, without undue experimentation. Such recombinant vectors
and packaging systems are within the scope of the invention.
[0095] The recombinant polynucleotides of the invention also
include nucleic acid sequences that encode for polypeptide analogs
or derivatives of the various polymerase and viral sequences, which
differ from naturally-occurring forms, e.g., deletion analogs that
contain less than all of the amino acids of the naturally-occurring
forms, substitution analogs that have one or more amino acids
replaced by other residues, and addition analogs that have one or
more amino acids added to the naturally-occurring sequence. These
various analogs share some or all of the biological properties of
the polymerase or viral sequences or polypeptides from which they
are derived. For example, but not limited to, catalyzing a DNA
template-directed RNA polymerization reaction, transcriptionally
regulating an operationally linked encoding sequence, forming a
psRNAV replication complex, transcribing a psRNAV "antigenome",
forming a psRNAV particle, and the like, as appropriate. One
skilled in the art will be able to design suitable analogs and to
test such analogs for biological activity using in vitro assay
systems.
[0096] In certain preferred embodiments, conservative amino acid
substitutions will be made. Conservative amino acid substitutions
include, but are not limited to, a change in which a given amino
acid may be replaced, for example, by a residue having similar
physiochemical or biochemical characteristics. Examples of such
conservative substitutions include, but are not limited to,
substitution of one aliphatic residue for another, such as lie,
Val, Leu, or Ala for one another, substitutions of one polar
residue for another, such as between Lys and Arg, Glu and Asp, or
Gin and Asn; or substitutions of one aromatic residue for another,
such as Phe, Trp, or Tyr for one another. Other conservative
substitutions, e.g., involving substitutions of entire regions
having similar hydrophobicity characteristics, are well known. See
Biochemistry: A Problems Approach, (Wood, W. B., Wilson, J. H.,
Benbow, R. M., and Hood, L. E., eds.) Benjamin/Cummings Publishing
Co., Inc., Menlo Park, Calif. (1981), page 14-15.
[0097] In certain embodiments, the analogs will be 70%, 75%, 80%,
85%, 90%, 95%, or 99% identical or homologous to the
naturally-occurring consensus sequences. As would be understood in
the art, percent identity involves the relatedness between amino
acid or nucleic acid sequences. One determines the percent of
identical matches between two or more sequences with gap alignments
that are addressed by a particular method. The percent identity may
be determined by visual inspection and/or mathematical calculation.
Alternatively, the percent identity of two nucleic acid sequences
can be determined by comparing sequence information using the GAP
computer program, version 6.0 described by Devereux et al. (Nucl.
Acids Res. 12:387, 1984) and available from the University of
Wisconsin Genetics Computer Group (UWGCG). The preferred default
parameters for the GAP program include: (1) a unitary comparison
matrix (containing a value of 1 for identities and 0 for
non-identities) for nucleotides, and the weighted comparison matrix
of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as
described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence
and Structure, National Biomedical Research Foundation, pp.
353-358, 1979; (2) a penalty of 3.0 for each gap and an additional
0.10 penalty for each symbol in each gap; and (3) no penalty for
end gaps. Other programs used by one skilled in the art of sequence
comparison may also be used.
[0098] Methods for obtaining infectious replicon particles are also
provided. According to certain methods, mammalian or insect cells
are coinfected with the recombinant viruses which form a novel
packaging system of the invention. Any cell line that has been
certified for use by the Food and Drug Administration for vaccine
production may be used. Other cell lines shown to be restrictive
for MVA growth may also be good candidates for replicon particle
production, including, but not limited to, CHO, CHL, CV-1, 293,
HeLa, SW 839, PK(15), MDCK, RK13, RAB-9, SIRC, Balb3t3, FS-2, MIB,
SK 29 MEL 1, LC 5, 85 HG 66, U 138, C 8166, HUT 78, SY 9287, and
Vero cells, described, among other places, in references (43),
(44), and (46). Preferred mammalian cells include BHK-21 cells and
FRhL cells. (ATCC Nos. CCL-10 and CL-160, respectively).
[0099] The coinfected cells are incubated under appropriate
conditions for replicon particles to be generated. The skilled
artisan will appreciate that appropriate conditions may vary, for
example, from one cell line to another or one packaging system to
another. The skilled artisan will know, or can readily determine,
the appropriate conditions for generating replicon particles. Such
appropriate conditions may include, for example, the optimal
incubation temperatures and times, CO.sub.2 concentrations, growth
media, supplements, serum source and concentrations, inhibitors of
DNA replication, such as AraC, and the like.
[0100] In certain embodiments of the invention, a vector containing
the structural genes of a psRNAV and a replicon expression vector
can be delivered to a cell using plasmids in lieu of viral vectors.
The vectors are delivered to the cell using transfection technology
that is well known in the art. Transfection methods are described
in Sambrook et al., Molecular Cloning (1989) (38). Expression of
the psRNAV genes may be driven by pol I promoters, pol II
promoters, bacteriophage promoters, and/or other suitable
promoters. In certain embodiments, the psRNAV structural genes and
replicon are under the control of pol II promoters, which may or
may not be inducible promoters. In certain embodiments, the psRNAV
structural genes and replicon are under the control of
bacteriophage promoters, such as a T7 promoter. In this system, the
expression of the psRNAV genes is dependent on the expression of a
T7 polymerase. T7 polymerase may be expressed by a cotransfected
plasmid, or may be stably expressed by the host cell. With either
system, the expression plasmids are cotransfected into a packaging
cell line and the replicon particles are harvested from the cell
medium.
[0101] Once generated, the replicon particles are obtained using
procedures that are well-known in the art. Exemplary procedures
include, but are not limited to, centrifugation, including
sedimentation and isopycnic centrifugation, chromatography, and
precipitation methods, such as selective precipitation using
polyethylene glycol, NaCl, or the like.
[0102] Methods for titering replicon particles are also provided.
In certain embodiments, an MVA recombinant is used to deliver a
reporter gene to a suitable cell line. Coinfection of the cells
with replicon particles activates the reporter gene, and the
coinfected cells can be detected. In an exemplary embodiment, an
MVA recombinant is made such that it contains a defective VEE RNA
that encodes green fluorescent protein (GFP). Upon coinfection with
replicon particles, the VEE replicase-transcriptase complex
replicates and transcribes the "VEE-like" RNA, and GFP protein is
expressed. The GFP can then be detected, and the titer of replicon
particles determined. Suitable reporter genes include, but are not
limited to, green fluorescent protein (GFP), blue fluorescent
protein, yellow fluorescent protein, chloramphenicol
acetyl-transferase (CAT), luciferase, beta-galactosidase,
beta-glucoronidase. Detection methods include, but are not limited
to, fluorescence microscopy, chemiluminescence, antibody staining,
enzymatic analysis, and colorimetric staining.
[0103] The replicon particles formed by the methods of the
invention can be employed as therapeutic or prophylactic
immunogenic compositions, or as pharmaceutical formulations,
depending at least in part on the foreign polypeptide(s) encoded in
the replicon particles. The sequence encoding the at least one
foreign polypeptide can vary as desired. Depending on the
application of a particular replicon particle, the sequence
encoding the at least one foreign polypeptide may encode a
co-factor, cytokine (such as an interleukin), an epitope for a T
cell, including helper, inducer, cytotoxic, and suppressor T cells,
a restriction marker, an adjuvant, a polypeptide from a pathogenic
microorganism, cancer or tumor cells, allergens, amyloid peptide,
protein or other macromolecular components.
[0104] Exemplary pathogenic microorganisms include, but are not
limited to, viruses, bacteria, fungi, or parasitic microorganisms
which infect humans and non-human vertebrates.
[0105] Examples of such viruses include, but are not limited to,
Human immunodeficiency virus, Simian immunodeficiency virus,
Respiratory syncytial virus, Parainfluenza virus types 1-3, Herpes
simplex virus, Human cytomegalovirus, Hepatitis A virus, Hepatitis
B virus, Hepatitis C virus, Human papillomavirus, poliovirus,
rotavirus, caliciviruses, Measles virus, Mumps virus, Rubella
virus, adenovirus, rabies virus, canine distemper virus, rinderpest
virus, coronavirus, parvovirus, infectious rhinotracheitis viruses,
feline leukemia virus, feline infectious peritonitis virus, avian
infectious bursal disease virus, Newcastle disease virus, Marek's
disease virus, porcine respiratory and reproductive syndrome virus,
equine arteritis virus and various Encephalitis viruses.
[0106] Examples of such bacteria include, but are not limited to,
Haemophilus influenzae (both typable and nontypable), Haemophilus
somnus, Moraxella catarrhalis, Streptococcus pneumoniae,
Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus
faecalis, Helicobacter pylori, Neisseria meningitidis, Neisseria
gonorrhoeae, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia
psiftaci, Bordetella pertussis, Salmonella typhi, Salmonella
typhimurium, Salmonella choleraesuis, Escherichia coli, Shigella,
Vibrio cholerae, Corynebacterium diphtheriae, Mycobacterium
tuberculosis, Mycobacterium avium-Mycobacterium intracellulare
complex, Proteus mirabilis, Proteus vulgaris, Staphylococcus
aureus, Clostridium tetani, Leptospira interrogans, Borrelia
burgdorferi, Pasteurella haemolytica, Pasteurella multocida,
Actinobacillus pleuropneumoniae and Mycoplasma gallisepticum.
[0107] Examples of such fungi include, but are not limited to,
Aspergillis, Blastomyces, Candida, Coccidiodes, Cryptococcus and
Histoplasma. Examples of such parasites include, but are not
limited to, Leishmania major, Ascaris, Trichuris, Giardia,
Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma gondii and
Pneumocystis carinii.
[0108] Exemplary polypeptides from cancer or tumor cells include,
but are not limited to, prostate specific antigen,
carcino-embryonic antigen, MUC-1, Her2, CA-125 and MAGE-3.
Exemplary allergens include, but are not limited to, those
described in U.S. Pat. No. 5,830,877 and published International
Patent Application No. WO 99151259, which include pollen, insect
venoms, animal dander, fungal spores and drugs (such as
penicillin). Such components interfere with the production of IgE
antibodies, a known cause of allergic reactions.
[0109] In one embodiment, the foreign polypeptide is amyloid
peptide protein (APP) which has been implicated in diseases
referred to variously as Alzheimer.quadrature.s disease,
amyloidosis or amyloidogenic disease. The .beta.-amyloid peptide
(also referred to as A-beta peptide) is a 42 amino acid fragment of
APP, which is generated by processing of APP by the .beta. and
.gamma. secretase enzymes, and has the following sequence:
1 (SEQ ID NO: 1) Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val
His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly
Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala.
[0110] In some patients, the amyloid deposit takes the form of an
aggregated A-beta peptide. Surprisingly, it has now been found that
administration of isolated A-beta peptide induces an immune
response against the A-beta peptide component of an amyloid deposit
in a vertebrate host (See Published International Patent
Application No. WO 99/27944). Such A-beta peptides have also been
linked to unrelated moieties. Thus, the heterologous nucleotides
sequences of this invention include the expression of this A-beta
peptide, as well as fragments of A-beta peptide and antibodies to
A-beta peptide or fragments thereof. One such fragment of A-beta
peptide is the 28 amino acid peptide having the following sequence
(as disclosed in U.S. Pat. No. 4,666,829):
2 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys
(SEQ ID NO: 2) Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys.
[0111] Foreign polypeptide of some embodiments may also include a
sequence that is expressed as a single transcriptional unit.
However, additional monocistronic transcriptional units or
polycistronic transcriptional units may also be included. Use of
the additional monocistronic transcriptional units, and
polycistronic transcriptional units should permit the insertion of
more genetic information. Where, for example, a polycistronic
transcriptional unit is included, the sequence may further comprise
one or more ribosomal entry sites. Alternatively, the foreign
sequence may encode a polyprotein and a sufficient number of
proteases that cleaves the polyprotein to generate the individual
polypeptides of the polyprotein.
[0112] Those skilled in the art would readily recognize that the
replicon particles of the invention may be used alone or in
conjunction with pharmaceuticals, antigens, immunizing agents or
adjuvants, as vaccines in the prevention or amelioration of
disease. These active agents can be formulated and delivered by
conventional means, i.e. by using a diluent or pharmaceutically
acceptable carrier.
[0113] Accordingly, in further embodiments of this invention the
replicon particles may be employed in immunogenic compositions
comprising (i) at least one replicon particle and (ii) at least one
of a pharmaceutically acceptable buffer or diluent, adjuvant or
carrier. Preferably, these compositions have therapeutic and
prophylactic applications as immunogenic compositions in preventing
and/or ameliorating, for example, but without limitation,
infectious diseases, cancer and other malignant conditions,
allergic reactions, autoimmune conditions, and the like. In such
applications, an immunologically effective amount of at least one
replicon particle of the invention is employed to cause a
substantial reduction in the course of the disease, reaction,
condition, or malignancy.
[0114] Suitable pharmaceutically acceptable carriers and/or
diluents include any and all conventional solvents, dispersion
media, fillers, solid carriers, aqueous solutions, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like. The term "pharmaceutically
acceptable carrier" refers to a carrier that does not cause an
allergic reaction or other untoward effect in patients to whom it
is administered. Suitable pharmaceutically acceptable carriers
include, for example, one or more of water, saline,
phosphate-buffered saline, dextrose, glycerol, ethanol and the
like, as well as combinations thereof. Pharmaceutically acceptable
carriers may further comprise minor amounts of auxiliary substances
such as wetting or emulsifying agents, preservatives or buffers,
which enhance the shelf life or effectiveness of the composition.
The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, use thereof in immunogenic compositions of the present
invention is contemplated.
[0115] Administration of such immunogenic pharmaceutical
formulations may be by any conventional effective form, such as
intranasally, parenterally (e.g. by subcutaneous, intramuscular, or
intravenous injection), orally, or topically applied to mucosal
surface such as intranasal, oral, eye, lung, vaginal, or rectal
surface, such as by aerosol spray.
[0116] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, and the like.
[0117] The immunogenic compositions or pharmaceutical formulations
of the invention can include an adjuvant, including, but not
limited to, aluminum hydroxide; aluminum phosphate; Stimulon.TM.
QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, Mass.); MPL.TM.
(3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Mont.);
synthetic adjuvant RC-529 (an aminoalkyl glucosamine phasphate
derivative; Corixa Corp., Seattle, Wash.); IL-12 (Genetics
Institute, Cambridge, Mass.); GM-CSF (Immunex Corp., Seattle,
Wash.); N-acetyl-muramyl-L-threonyl-D-isoglutami- ne (thr-MDP);
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP); N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alan-
ine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine
(CGP 19835A, referred to a MTP-PE); and cholera toxin. Others which
may be used are non-toxic derivatives of cholera toxin, holotoxins
having reduced toxicity compared to wild-type cholera toxins,
including it's A subunit (for example, wherein glutamic acid at
amino acid position 29 is replaced by another amino acid,
preferably, a histidine in accordance with Published International
Patent Application No. WO 00/18434), and/or conjugates or
genetically engineered fusions of the at least one foreign
polypeptide with cholera toxin or its B subunit, procholeragenoid,
fungal polysaccharides.
[0118] One important aspect of the invention relates to a method
for inducing an immune response in a mammal or human host
comprising administering an immunogenic composition of the
invention to the host. Provided that an immunologically effective
amount of the immunogenic composition is administered, the host
will develop a desired immune response. The dosage amount can vary
depending upon specific circumstances, such as size (weight) and
the developmental state of the host individual. This amount can be
determined in routine trials by means, known to those skilled in
the art.
[0119] Certainly, the isolated foreign polypeptides produced using
the packaging systems and/or methods of the invention may be used
in forming subunit vaccines. They may also be used as antigens for
raising polyclonal or monoclonal antibodies and in immunoassays for
the detection of antibodies that are reactive with the foreign
polypeptide(s) of the invention. Immunoassays encompassed by the
present invention include, but are not limited to those described
in U.S. Pat. No. 4,367,110 (double monoclonal antibody sandwich
assay) and U.S. Pat. No. 4,452,901 (western blot). Other assays
include immunoprecipitation of labeled ligands and
immunocytochemistry, both in vitro and in vivo.
[0120] The invention also provides kits designed to expedite
performing the subject methods. Kits serve to expedite the
performance of the methods of interest by assembling two or more
components required for carrying out the methods. Kits preferably
contain components in pre-measured unit amounts to minimize the
need for measurements by end-users. Kits preferably include
instructions for performing one or more methods of the invention.
Preferably, the kit components are optimized to operate in
conjunction with one another.
[0121] Kits may also be used to generate the packaging systems
disclosed herein or to insert desired foreign genes into the psRNAV
replicon. Kits of the invention include kits that facilitate
titering replicon particles. The immunogenic compositions and
pharmaceutical formulations of the invention may be prepared using
the disclosed kits.
[0122] The invention, having been described above, may be better
understood by reference to examples. The following examples are
intended for illustration purposes only, and should not be
construed as limiting the scope of the invention in any way. While
MVA and VEE are used as exemplary poxvirus and positive-strand RNA
virus systems, respectively, the skilled artisan will appreciate
that other poxviruses and positive-strand RNA viruses may be used
interchangeably without undue experimentation. Thus, the use of
other poxviruses and/or alphaviruses are contemplated and are
within the intended scope of the invention. Although the wild-type
strains of vaccinia virus could also be used to package VRPs, their
cytopathic effect and unrestricted growth in a broad range of cells
would limit their utility. Other host-range defective mutants of
vaccinia virus including the NYVAC strain or naturally occurring
poxviruses with limited host-range (e.g., avipoxvirus,
parapoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, or
entomopoxviruses) could also be used as packaging vectors. Other
positive-strand RNA viruses that may be used included, but are not
limited to, Rubella, Hepatitis C virus, Dengue virus, Coronavirus,
and the like.
EXAMPLES
Example 1
Materials and Methods
[0123] 1A Development of a Recombinant MVA Vector Capable of
Expressing Abundant T7 RNA Polymerase Early in Infection.
[0124] A stock of MVA (24), (23), (25), obtained from Dr. Bernard
Moss (NIAID), was plaque purified and amplified on certified chick
embryo fibroblasts (CEF; SPAFAS) in minimal essential media (Life
Technologies) supplemented with 10% fetal bovine serum (Life
Technologies). This virus was utilized as the parent for insertion
of all foreign genes and expression cassettes.
[0125] A recombinant MVA expression vector (MVGKT7) was engineered
to express abundant amounts of the bacteriophage T7 RNA polymerase
early in infection (FIG. 2). It was necessary to express abundant
levels of T7 polymerase early in infection because, as demonstrated
below, VEE coinfection limits expression from vaccinia late
promoters. The T7 RNA polymerase gene was excised from pT7-Neo
(gift of Dr. S. Lee, Wyeth Lederle Vaccines) as a BamHI fragment
and subcloned into the Bg/II site of pSC65 ((9); obtained from Dr.
Bernard Moss), to generate pGK16.2 (see FIG. 5). The skilled
artisan will understand T7 can come from other sources. This
plasmid contains flanking sequences for homologous recombination
into the thymidine kinase locus of MVA, a lac-Z marker gene, and a
synthetic early/late vaccinia virus promoter (9) regulating the
transcription of the T7 RNA polymerase gene. The recombinant virus,
MVGKT7, was produced using methods described previously (36).
Briefly, CEF were infected with MVA at a multiplicity of infection
(MOI) equal to 0.5 plaque forming units (PFU) per cell, and
subsequently transfected with pGK16.2 using DOTAP transfection
reagent (Boehringer Mannheim). Recombinant viruses were
plaque-purified three times consecutively on CEF using a
5-Bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-gal)
colorimetric plaque assay (8), (22). The purity and stability of
the recombinant virus was assessed by immunostaining with a rabbit
polyclonal anti-T7 polymerase antibody (gift of Dr. S. Lee, Wyeth
Lederle Vaccines) and a polyclonal antiserum raised against
vaccinia virus (BioGenesis). The skilled artisan will appreciate
that polyclonal anti-T7 polymerase antiserum is easily generated
using commercially available T7 polymerase in conventional serum
preparation methods. Detailed descriptions of such methods may be
found, among other places, in Harlow and Lane, eds. Antibodies, A
Laboratory Manual, Cold Spring Harbor Press, 1988.
[0126] 1 B. Development of an Inducible MVA-Based VRP Packaging
System.
[0127] An expression vector plasmid, pMCO3, used for insertion of
foreign DNA into deletion III of MVA (obtained from B. Moss, (7))
was modified by digesting with BamHI and religating (see FIG. 6).
This removed the 7.5K promoter used to transcribe the marker gene
.beta.-glucuronidase (GUS) and moved the synthetic early/late
promoter, used to transcribe foreign genes, upstream of GUS. The
modified plasmid, pDF17, contains a unique PstI site upstream of
the synthetic early/late promoter-GUS gene. The defective-helper
gene cassettes were derived from plasmids
pV3014.DELTA.520-7505.DELTA.8495-11229 and
pV3014.DELTA.520-7505.DELTA.75- 65-8386 herein referred to as pVcap
and pVgP, respectively (obtained from AlphaVax, (27)). Both helpers
were originally isolated from V3014, a laboratory-derived, highly
attenuated VEE mutant that contains a mutation in E1 (A272T) and
two in E2 (E209K, 1239N), yet is immunogenic and has been used as
an expression vector (10), (12). Plasmids pVcap and pVgP were
digested with EcoRI/PstI or HindIII/EcoRI, respectively, to excise
the T7 promoter-helper gene cassettes (see FIG. 7). The ends of
both cassettes were modified to contain PstI-compatible cohesive
ends, and were then cloned individually into the PstI site of
pDF17. The resultant plasmids contain either the capsid defective
helper (pGK51) or the glycoprotein defective-helper (pGK53)
expression cassettes under the regulation of T7 promoters (see FIG.
7). Recombinant MVA vectors were produced by transfection of pGK51
into MVGKT7-infected cells to yield MVGKT7/DHCap (FIG. 8A), and
transfection of pGK53 into MVA-infected cells to yield MVA/DHgP.
Both recombinant viruses were selected using a colorimetric plaque
assay based on GUS gene expression (7). These viruses express
defective-helper VEE RNAs that are not translated into capsid or
glycoproteins, unless they are replicated by the VEE replicase.
Thus, expression of the helpers is referred to as "inducible".
[0128] The second step in the development of the inducible
MVA-based VRP packaging system was to engineer a recombinant MVA
vector capable of expressing a full-length VEE replicon under the
control of a T7 promoter. As shown in FIG. 9, several modifications
were made to pLW17 (an expression vector that enables insertion of
foreign genes into deletion II of MVA; obtained from Dr. B. Moss;
see FIG. 18; SEQ ID NO: 3) to allow for cloning of replicon cDNAs.
First, pLW17 was digested with SmaI and PstI to remove the vaccinia
virus H5R promoter used for foreign gene expression, and a unique
NotI restriction site was inserted in its place to yield pDF13 (See
FIG. 9). To aid cloning into the final transfer vector, a unique
XbaI site present in one of the homologous flanking regions in
pDF13 was ablated by digestion, filled-in with T4 DNA polymerase
and religated. The resultant plasmid, pDF49, was digested with
NotI, and a polylinker containing unique NotI, XbaI, Sse83871,
SmaI, and SalI restriction sites was inserted to yield pDF51 (See
FIG. 9). A vaccinia virus intermediate promoter (G8R)-lacZ
expression cassette was derived from p30/300 (obtained from Dr. B.
Moss; (1)) and inserted into the SmaI site present in the
polylinker of pDF51 to allow colorimetric selection of recombinant
viruses. The resultant plasmid (pGK61) contains two regions of
homology with MVA for insertion into deletion II, a lacZ
colorimetric marker gene, and three unique restriction sites
(Sse83871, XbaI and NotI) for directional cloning of VEE replicon
cDNAs (See FIG. 9).
[0129] A VEE replicon vector capable of expressing the green
fluorescent protein (GFP) gene was constructed by removing the GFP
gene from pEGFP-N3 (Clontech; Palo Alto, Calif.) as a SmaI-NotI
fragment, filling-in staggered ends with T4 DNA polymerase, and
inserting into the EcoRV site of pVR3 (a derivative of pVR200
obtained from AlphaVax), (See FIG. 10). The resultant expression
plasmid, pVRGFP (provided by Dr. Larry Smith; WyethLederle
Vaccines), was digested with XbaI and NotI to excise the entire
replicon genome-GFP preceded by a T7 promoter (see FIG. 10). The T7
promoter-linked replicon-GFP fragment was subsequently inserted
into pGK61 to yield pGK63 (See FIG. 11). This plasmid was then
transfected into MVA/DHgP-infected cells to yield MVANEEGFP/DHgP
(see FIG. 8A), a recombinant MVA virus that contains both a VEE
replicon-GFP cDNA and the glycoprotein helper gene under the
transcriptional regulation of T7 promoters
[0130] 1 C. Development of a Constitutive MVA-Based VRP Packaging
System.
[0131] The polymerase chain reaction (PCR) was used to amplify the
open reading frames (ORFs) of the capsid and E3/E2/6K/E1
polyprotein cassette from pVRCap and pVRgP, respectively. These
fragments were then cloned into pDF33 (a deletion III MVA
expression vector that was derived from pMCO3, (7)) (see FIGS. 6
and 12). The resultant plasmids pGK64 and pGK65 contain the capsid
or E3/E2/6K/E1 polyprotein ORFs, respectively, under the regulation
of vaccinia virus synthetic early/late promoters. A recombinant MVA
virus capable of expressing the T7 RNA polymerase and the VEE
glycoproteins was produced by transfection of pGK65 into
MVGKT7-infected CEF using the methods described above. The
resultant virus (MVGKT7/gP) constitutively expresses T7 RNA
polymerase and the VEE spike glycoproteins (FIG. 8B).
[0132] A separate recombinant MVA virus capable of transcribing the
capsid gene and a VEE replicon RNA was engineered in two steps.
First, a recombinant MVA virus was isolated from MVA-infected cells
that had been transfected with pGK64. This virus (MVA/cap) contains
an expression cassette comprising the VEE capsid gene under the
control of the synthetic early/late promoter inserted into deletion
III of MVA. Second, pGK63 (from Example 1B, above) was transfected
into MVA/cap-infected cells to yield MVA/VEEGFP/cap (FIG. 8B). This
virus expresses the VEE capsid gene and also contains a T7
promoter-regulated replicon-GFP expression cassette
[0133] 1D. Characterization of VRP-Packaging MVAs.
[0134] Recombinant plasmids were sequenced using dye terminator
cycle sequencing and the 377 ABI DNA sequencer (Applied
Biosystems). Recombinant MVA viruses were checked for purity by PCR
analysis. Expression of the E1 and E2 glycoproteins and capsid was
assayed by Western blot analyses. Approximately 2.times.10.sup.6
baby hamster kidney (BHK-21) cells were infected with recombinant
MVA viruses at a multiplicity of infection equivalent to 10 PFU per
cell, and incubated at 37.degree. C. for 24 hr. Subsequently, cells
were boiled in SDS disruption buffer (0.05 M Tris, 4% SDS, 4%
beta-mercaptoethanol, 10% glycerol, 0.1% bromophenol blue) and an
aliquot was analyzed by immunoblotting using mouse hyperimmune
anti-VEE sera (ATCC, Manassas, Va.) at a 1:1,000 dilution.
Immunoblots were developed using a rabbit anti-mouse IgG conjugated
to alkaline phosphatase secondary antibody (Life Technologies) and
the Western Blue substrate (Promega, Madison, Wis.). Expression of
the VEE structural proteins was also analyzed in BHK-21 cells that
were co-electroporated with in vitro transcribed VEE replicon-GFP
RNA, gP helper and capsid helper RNAs as previously described (19),
(27). (See FIG. 16).
[0135] 1 E. Production of VEE Replicon Particles (VRP)
[0136] A VEE replicon particle, referred to as VRP/GFP, that
expresses the GFP gene under the regulation of the subgenomic
promoter was obtained by co-infecting BHK-21 cells with
MVGKT7/DHCap and MVA/VEEGFP/DHgP (an exemplary inducible system;
see FIG. 4) or MVGKT7/gP and MVANEEGFP/cap (an exemplary
constitutive system; see FIG. 3). Typically, recombinant viruses
were adsorbed in a minimal volume of inocula for 1 h with rocking
at room temperature. Infected cells were washed twice with PBS, and
incubated in modified Eagle's medium (MEM) containing 10% fetal
bovine serum (FBS). Replicon particles were harvested from
infected-cell media after 24 hpi using centrifugation at
3,000.times.g for 10 min. Serial dilutions of VRP/GFP preparations
were assayed on fresh BHK-21 cells and the titer, expressed as
IU/ml, was determined by counting the total number of fluorescent
cells per well at an appropriate dilution.
[0137] The titer of the recombinant MVA helper viruses in the VRP
preparations was determined by infecting CEF with serially-diluted
infected-cell media, fixing cells with 2% formaldehyde at 48 hpi.,
and subsequently immunostaining monolayers with anti-vaccinia virus
antisera (BioGenesis) followed by a horseradish
peroxidase-conjugated anti-rabbit IgG secondary antibody (Life
Technologies), and staining with the AEC Peroxidase Substrate Kit
(Enzo). The titers of recombinant MVA helper viruses were expressed
as PFU/ml. Possible contaminating replication-competent VEE,
resulting from recombination between helper RNAs and the replicon,
was assayed by a standard plaque assay on Vero cells after three
serial undiluted passages in naive Vero cells. This assay
differentiates between replicon particles and replication-competent
virus on the basis of viral growth after the second passage.
Example 2
Characterization of MVA and VEE Coinfection
[0138] To evaluate whether VEE coinfection of MVA infected cells
would affect poxvirus early and/or late gene expression, two
recombinant MVA viruses that express lacZ under the control of
either a viral early promoter (MVA/7.5KlacZ) or a late promoter
(MVA/11KlacZ) were used to infect cells with or without VEE
coinfection. BHK-21 cells were infected with 10 PFU of MVA/7.5KlacZ
or MVA11 KlacZ per cell alone or with 10 IU per cell of VRP/GFP.
Cells were harvested at 24 hpi, and assayed for
.beta.-galactosidase activity, a measure of poxvirus gene
expression (see FIG. 13). The levels of .beta.-galactosidase
detected in samples 1 and 3 of FIG. 13 are indicative of normal
early and late gene expression during MVA infection, respectively.
Addition of cytosine-beta-D-arabinofuranosid- e (AraC), a drug that
blocks MVA DNA replication, shows that late but not early genes are
inhibited (FIG. 13, compare sample 2 to 4). Coinfection of cells
with MVA/11KlacZ and VRP/GFP shows that VEE replication has an
effect on late gene expression that is similar to AraC.
.beta.-galactosidase expression was reduced in coinfected cells by
nearly 95% (FIG. 13, compare samples 3,4, and 7). This indicates
that MVA DNA replication and/or late gene transcription is
inhibited by VEE replication. MVA early gene expression was also
reduced by coinfecting with VRP/GFP, albeit to a lesser extent than
late gene expression (FIG. 13, compare samples 1 to 6).
[0139] A similar experiment was conducted to determine the effects
of MVA infection on VEE replication. (See Table 1). BHK-21 cells
were infected with VRP/GFP alone or together with MVA. At 24 hpi,
cells were analyzed for expression of GFP by flow cytometry using a
FACScan (Beckton Dickinson) and Cell Quest 3.1 software. The
intensity of GFP fluorescence is directly proportional to the level
of VEE subgenomic promoter transcription. Surprisingly, cells that
were co-infected with VRPIGFP and MVA expressed at least 50-60%
more GFP than cells infected with VRP/GFP alone. This suggests that
one or more MVA gene products appear to facilitate or stimulate the
replication and/or transcription of VEE. (See Table 1),
[0140] The results shown in FIG. 13 and Table 1 suggested that: 1)
vaccinia virus strong early promoters (e.g. a synthetic early/late
(9), H5R (48), Pse1 (47)) could be used to express the VEE
replicon, and structural genes, 2) the growth of the VRP-packaging
MVAs would be severely impaired during VEE replicon packaging,
thereby further reducing the risk of adventitious contamination of
VRP with MVA, and 3) in the presence of an ongoing MVA infection,
VEE replication and subsequent particle formation may be
enhanced.
Example 3
Characterization of an Inducible MVA-Based VRP Packaging System
[0141] The titers obtained by coinfection with MVA/VEEGFP/DHgP
(IMVA1) and MVGKT7/DHCap (IMVA2) (see FIG. 4) were compared to
those produced with the standard split-helper RNA transfection
method. BHK-21 cells were chosen as the cell substrate for
packaging because they have been shown to produce the highest
titers of VRP. However, BHK-21 cells are not appropriate for mass
production of VRP using the MVA-based VRP-packaging systems because
they are fully permissive for MVA growth (6), (13), (4). BHK-21
cells were infected with two recombinant MVA vectors constituting
the inducible packaging system at an MOI of 1, 10 or 20 total
PFU/cell. Alternatively, the cells were co-electroporated with VEE
replicon-GFP RNA, capsid DH RNA, and gP DH RNA synthesized in vitro
by T7 RNA polymerase.
[0142] Media from infected and electroporated cells were harvested
at 24 h and titered on fresh BHK-21 cells. Additionally, some of
the original infected and electroporated cells were trypsinized and
counted at the time of harvest to calculate VRP production on a
per-cell basis. The results showed that the inducible MVA-based VRP
packaging system produced on the average between 20-60 VRP/cell,
whereas the RNA transfection method yielded approximately 15
VRP/cell (see FIG. 14).
[0143] Although the MVA-based VRP packaging system produced more
VRP than the split-helper RNA transfection method, the biosafety
issues remained since replication-competent viruses might be
generated during the replicon packaging process. This is due to the
fact that both the inducible MVA-based VRP-packaging system and the
in vitro RNA transfection method employ DH RNAs that are capable of
recombining with the replicon.
Example 4
Characterization of a Constitutive MVA-Based VRP-Packaging
System
[0144] The recombination rate between RNA species during an
alphavirus infection has been estimated to be 10.sup.-6 per
replication cycle (3). Furthermore, there appears to be a direct
correlation between the length of replication sequences on the ends
of the helper RNAs and the likelihood of recombination with the
replicon RNA (27). To generate a replication-competent virus using
a split-helper expression system two recombination events are
required, significantly reducing the probability from 10.sup.-6 to
10.sup.-12. However, single-recombination events lead to a
significant proportion of VRPs that have genomes capable of
expressing one, but not both, of the structural genes in addition
to the foreign gene. These particles could theoretically preclude
repeated immunizations with recombinant VRPs by inducing a
vector-specific immune response. In theory, if helper genes could
be expressed as individual mRNAs that lack VEE-specific regulatory
elements, instead of DH RNAs, the likelihood of producing
replication-competent VEE by recombination would be further
decreased. Furthermore, if a single recombination event took place
between a helper mRNA and the VEE replicon, then the probability of
expressing the helper gene would be infinitesimal since it lacks a
subgenomic promoter.
[0145] We observed in preliminary experiments that constitutive
co-expression of the VEE capsid and glycoproteins by the same
vector inhibited growth of recombinant MVAs. By inserting the
capsid and glycoproteins genes into separate recombinant vectors,
stable recombinant MVA viruses were isolated that grew to normal
titers (>10.sup.8 PFU/ml). Initial experiments were performed to
determine the titers of VRP/GFP that are produced in BHK-21 cells
co-infected with MVA/VEEGFP/cap (CMVA2) and MVGKT7/gP (CMVA1;
exemplary constitutive system; see FIG. 8B), and to compare them to
titers obtained by the split-helper RNA transfection method.
Infections were performed with MOIs equivalent to 1, 10, or 20 PFU
of total virus per cell. The media from infected and transfected
cells were harvested at 24 h, and VRP/GFP titers were determined.
Coinfection with the constitutive recombinant MVA viruses yielded
titers as high as 2.times.10.sup.8 IU per 1.times.10.sup.6 cells,
or approximately 190 VRP/GFP per cell (see FIG. 15). This system
regularly yielded higher VRP titers than the DH RNA transfection
method.
[0146] VRPs were also produced by providing a recombinant replicon
and helper functions as transfected plasmids. BHK-21 cells were
transfected with the following combination of plasmids:
pGK63+pGK51+pGK53 or pGK63+pGK64+pGK65. Subsequently, transfected
cells were infected with MVGKT7. The media from
transfected/infected cells was titered for VRP/GFP at 24 h. The
titers of VRP/GFP were comparable to those obtained by
co-transfection of a VEE replicon-GFP RNA and the two split-helper
RNAs (data not shown).
Example 5
Comparison of Structural Protein Expression Using MVA-Based VRP
Packaging Systems and RNA Electroporation.
[0147] To determine the amounts of alphavirus capsid protein and
glycoprotein produced by the MVA-based VRP packaging systems and
the RNA electroporation method, lysates from infected and
electroporated cells were analyzed by immunobloting. Twenty-four
hpi, cell lysates were prepared and probed with anti-VEE antiserum.
As shown in FIG. 16A, the expression of capsid (Cap), E1, and E2
(gP), is slightly higher in cells infected with the constitutive
MVA-based VRP-packaging system than with the inducible MVA-based
VRP-packaging system or RNA electroporation. FIG. 16A also
illustrates that expression of the VEE structural genes is possible
only when cells are coinfected with both of the exemplary inducible
recombinant MVA viruses. In contrast, the respective structural
genes in the individual constitutive recombinant MVA viruses are
expressed independent of coinfection (see FIG. 16A, compare lanes
3, 4, and 5 to lanes 7, 8, and 9). Lastly, infected- and
transfected-cell media were titered for the presence of VRP/GFP
(FIG. 16B). The results indicate that there is a direct correlation
between VRP titers and the amount of structural proteins produced
by each packaging system.
Example 6
VRP Packaging in Cells that are Restrictive for MVA Growth.
[0148] Since BHK-21 cells are fully permissive for MVA growth, they
are not an ideal cell line for MVA-based VRP packaging systems. A
panel of cells that were restrictive for MVA growth was tested in a
VRP packaging experiment. Equivalent numbers of cells were infected
with 10 PFU/cell of MVA/VEEGFP/cap and MVGKT7/gP (constitutive
system) and incubated for 24 h. Media from infected-cells were
harvested and VRP/GFP titers were determined on fresh BHK-21 cells.
As shown in FIG. 17 two human cell lines (MRC-5 and WI38), one
hamster cell line (CHO), and a nonhuman primate cell line (Vero)
yielded VRP titers that were significantly lower than those
obtained with BHK-21 cells. Surprisingly, fetal Rhesus monkey lung
(FRhL) cells produced VRP/GFP titers as high as BHK-21.
Example 7
The Growth of MVA is Severely Inhibited During VRP-Packaging
[0149] One potential limitation to using the MVA-based VRP
packaging systems is that VRP preparations could be contaminated
with MVA recombinants even though VRPs bud from the cell membrane
while most of the MVA virus remains cell-associated. As shown in
Example 2, MVA late gene expression is inhibited by VEE
replication. To measure the level of MVA growth inhibition, BHK-21
cells (permissive for MVA growth) or FRhL cells (restrictive for
MVA growth) were infected with MVGKT7/gP alone or together with
MVA/VEEGFP/cap. Infection with MVGKT7/gP alone would not result in
either VEE replication or production of VRP. Coinfection with
MVA/VEEGFP/cap, however, results in VEE replicon replication and
VRP production (see FIG. 15). The media from single-infected and
coinfected cells were harvested at 24 hpi, and recombinant MVA
viruses were titered on CEF. The host-range growth restriction is
clearly shown in FRhL cells infected with MVGKT7/gP alone (Table
2). For example, in the absence of VEE replication, the average
PFU/cell produced in BHK-21 and FRhL cells was 435 and 0.6
PFU/cell, respectively. When coinfected with MVAGKT7/gP and
MVAVEEGFP/cap, the average PFU/cell was decreased to 1.1 in BHK-21
cells (normally permissive), and to 0.007 in FRhL cells. Thus,
consistent with the results obtained in Example 2, there is a great
reduction in MVA growth during VEE replication.
Example 8
Method for Titering VRPs Using an MVA Indicator Virus
[0150] This section describes a method for titering VEE replicon
particles (VRPs) that makes use of recombinant MVA viruses similar
to those used in packaging these particles. Briefly, an MVA
recombinant is used to deliver a reporter gene to a suitable cell
line growing in culture. Coinfection of these cells with replicon
particles activates the reporter gene of the MVA indicator virus so
that the infected cells can be detected, counted, and thus a titer
for the preparation can be determined.
[0151] This titering system fulfills an important need in the use
of VRPs for research, gene expression, and vaccine production.
Currently the only practical method of titering a VRP preparation
is by immunohistochemistry, using an antibody directed against the
foreign protein or a tag engineered on the foreign protein encoded
by the VRP. However, antibodies are not always available for the
specific gene products, and making antibodies is a lengthy process
requiring the isolation and purification of immunogens.
[0152] This assay makes use of the fact that all functional
replicon particles encode VEE non-structural proteins (nsPs) that
replicate and transcribe VEE RNA. By assaying for functional nsP,
this assay is capable of measuring titers of VRPs independently of
the foreign gene products that they express. This makes comparisons
between VRP preparations more accurate, especially in cases where
the sensitivity of detection by immuno-detection is significantly
different between preparations.
[0153] The plasmid transfer vector for construction of such an MVA
virus indicator (p21Q4) is diagrammed in FIG. 19. It consists of
two gene cassettes, a gene coding for a defective VEE RNA encoding
green fluorescent protein (GFP) under control of the phage T7
transcriptional promoter, and the glucuronidase (gus) gene under
control of a vaccinia synthetic early-late promoter, as a
selectable marker. The structure of the defective VEE RNA is
similar to the "replicon-like helper RNA" referred to above. Thus,
the RNA that is produced has 5' and 3' ends identical to that of
VEE viral RNA, 90% of the region of VEE coding for nsPs is deleted,
and the VEE sub-genomic promoter directs the synthesis of a protein
product. However, instead of coding for either VEE glycoproteins or
VEE capsid protein, this RNA encodes the GFP reporter protein. The
gus gene serves to enable isolation of recombinant viruses. These
two gene cassettes in p2104 are flanked by MVA sequences that
direct the recombination of this plasmid into deletion III of MVA.
The p2104 plasmid is recombined into deletion III of an MVA that
already encodes T7 RNA polymerase under a synthetic early/late
promoter at the thymidine kinase locus (MVGKT7; see FIG. 2). The
resulting virus (IMVA3, FIG. 20) is used as an indicator in the
titration assay.
[0154] The universal replicon titering system is depicted in FIG.
21. A cell line that is non-permissive for MVA but permissive for
VEE replication is used (e.g. Vero). Cells are infected with the
IMVA3 indicator virus at a multiplicity of 5 to ensure that all
cells in the culture are infected. This virus synthesizes T7 RNA
polymerase, which recognizes the T7 promoter (T7 Pr) and
transcribes the defective "VEE-like" RNA coding for GFP (nsP-GFP).
Since the start of translation is approximately 500 bases from the
5' end of this RNA and since several stop codons are positioned
before the GFP open reading frame, GFP is not translated from this
RNA. The MVA indicator-infected cultures are co-infected with
serial dilutions of a VRP preparation of unknown titer. The VRP
delivers, to the cytoplasm of the MVA indicator-infected cell, its
replicon RNA which is immediately translated to produce the VEE
replicase-transcriptase complex encoded by the nsP gene. The VEE
replicase initiates replication of the defective GFP RNA, and also
transcribes the subgenomic promoter present on the minus-strand
(antisense) replication intermediate. This results in the synthesis
of large quantities of mRNA encoding GFP (subgenomic GFP RNA)
Translation of the GFP mRNA causes the cell to fluoresce under UV
light. The fluorescent cells are counted and the titer of the
original stock of VRPs is determined by extrapolation.
[0155] The IMVA3 indicator virus was tested for its ability to
titer a VEE replicon particle expressing the herpes simplex virus
glycoprotein D (VRPgD). Confluent cultures of Vero cells were
infected with IMVA3 at a MOI equivalent to 10 PFU/cell, or with
IMVA3 at an MOI of 10 PFU/cell plus serial dilutions of a VRPgD
preparation of unknown titer. Cells were fixed at 24 hpi with 2%
formaldehyde and visualized using fluorescent microscopy (FIG. 23).
The results indicate that GFP is not expressed unless cells are
coinfected with both a replicon particle and the IMVA3 indicator
virus. Panel C of FIG. 23 shows a representative field in the VRPgD
dilution series.
[0156] Interference between VEE replication and MVA gene expression
is not an issue since we have already established, with data
presented in Example 2 (Table 1), that MVA infection actually
enhances VEE replication. We also showed that it is only late MVA
gene expression that is inhibited by VEE (FIG. 13). Accordingly,
this assay makes use of a vaccinia promoter that functions early as
well as late.
[0157] A universal assay for Sindbis virus replicons has been
previously described (53). This system uses a defective RNA
encoding a reporter gene whose transcription was under the control
of a cellular RNA polymerase II promoter. In order to use this
system, it would first be necessary to isolate stable cell lines
expressing the transcript. The isolation of stable mammalian cell
lines is a time consuming and laborious undertaking. An MVA-based
reporter system significantly reduces the labor in setting up such
an assay.
Example 9
Method for Amplifying Replicon Particles
[0158] In addition to its utility as a means of producing
alphavirus replicon particles de novo, it is also possible to
amplify alphavirus replicon particles using the MVA expression
vectors. To amplify a preparation of alphavirus replicon particles
one coinfects a cell line (eg. fetal rhesus lung, or FRhL) with the
replicon particle preparation at a low MOI (1-5 IU/cell) and a
higher MOI of MVA recombinant viruses (5-20 PFU/cell) that express
the structural genes needed for packaging. At 24-48 hpi, the medium
is collected and newly packaged replicon particles are purified as
described in Example 1.
[0159] Provided below is a list of references which are expressly
incorporated herein for any purpose.
[0160] 1. Baldick, C. J., J. G. Keck, and B. Moss. 1992. Mutational
analysis of the core, spacer and initiator regions of vaccinia
virus intermediate class promoters. J. Virol. 66:4710-4719.
[0161] 2. Barrett, N., A. Mitterer, W. Mundt, J. Eibl, M. Eibl, R.
C. Gallo, B. Moss, and F. Dorner. 1989. Large-scale production and
purification of a vaccinia recombinant-derived HIV-1 gp160 and
analysis of its immunogenicity. AIDS Res. Human Retroviruses.
5:159-171.
[0162] 3. Berglund, P., M. Sjoberg, H. Garoff, G. J. Atkins, B. J.
Sheahan, and P. Liljestrom. 1993. Semliki Forest virus expression
system: production of conditionally infectious recombinant
particles. Bio/Technology. 11:916-920.
[0163] 4. Blanchard, T. J., A. Alcami, P. Andrea, and G. L. Smith.
1998. Modified vaccinia virus Ankara undergoes limited replication
in human cells and lacks several immunomodulatory proteins:
implications for use as a human vaccine. Journal of General
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[0220] Although the invention has been described with reference to
various applications, methods, and compositions, it will be
appreciated that various changes and modifications may be made
without departing from the invention.
Sequence CWU 1
1
3 1 42 PRT Homo sapiens 1 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val
Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly
Val Val Ile Ala 35 40 2 28 PRT Homo sapiens 2 Asp Ala Glu Phe Arg
His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe
Phe Ala Glu Asp Val Gly Ser Asn Lys 20 25 3 4082 DNA Human
immunodeficiency virus Description of Artificial Sequence vector
pLW17 3 cctcctgaaa aactggaatt taatacacca tttgtgttca tcatcagaca
tgatattact 60 ggatttatat tgtttatggg taaggtagaa tctccttaat
atgggtacgg tgtaaggaat 120 cattatttta tttatattga tgggtacgtg
aaatctgaat tttcttaata aatattattt 180 ttattaaatg tgtatatgtt
gttttgcgat agccatgtat ctactaatca gatctattag 240 agatattatt
aattctggtg caatatgaca aaaattatac actaattagc gtctcgtttc 300
agacatggat ctgtcacgaa ttaatacttg gaagtctaag cagctgaaaa gctttctctc
360 tagcaaagat gcatttaagg cggatgtcca tggacatagt gccttgtatt
atgcaatagc 420 tgataataac gtgcgtctag tatgtacgtt gttgaacgct
ggagcattga aaaatcttct 480 agagaatgaa tttccattac atcaggcagc
cacattggaa gataccaaaa tagtaaagat 540 tttgctattc agtggactgg
atgattcgag gtacccgggg atcctctaga gtcaacctta 600 tttatgatta
tttctcgctt tcaatttaac acaaccctca agaacctttg tatttatttt 660
caatttttag ctgcaggtgg atgcgatcat gacgtcctct gcaatggata acaatgaacc
720 taaagtacta gaaatggtat atgatgctac aattttaccc gaaggtagta
gcatggattg 780 tataaacaga cacatcaata tgtgtataca acgcacctat
agttctagta taattgccat 840 attggataga ttcctaatga tgaacaagga
tgaactaaat aatacacagt gtcatataat 900 taaagaattt atgacatacg
aacaaatggc gattgaccat tatggagaat atgtaaacgc 960 tattctatat
caaattcgta aaagacctaa tcaacatcac accattaatc tgtttaaaaa 1020
aataaaaaga acccggtatg acacttttaa agtggatccc gtagaattcg taaaaaaagt
1080 tatcggattt gtatctatct tgaacaaata taaaccggtt tatagttacg
tcctgtacga 1140 gaacgtcctg tacgatgagt tcaaatgttt cattgactac
gtggaaacta agtatttcta 1200 aaattaatga tgcattaatt tttgtattga
ttctcaatcc taaaaactaa aatatgaata 1260 agtattaaac atagcggtgt
actaattgat ttaacataaa aaatagttgt taactaatca 1320 tgaggactct
acttattaga tatattcttt ggagaaatga caacgatcaa accgggcatg 1380
caagcttgtc tccctatagt gagtcgtatt agagcttggc gtaatcatgg tcatagctgt
1440 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
ggaagcataa 1500 agtgtaaagc ctggggtgcc taatgagtga gctaactcac
attaattgcg ttgcgctcac 1560 tgcccgcttt cgagtcggga aacctgtcgt
gccagctgca ttaatgaatc ggccaacgcg 1620 cggggagagg cggtttgcgt
attgggcgct cttccgcttc ctcgctcact gactcgctgc 1680 gctcggtcgt
tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 1740
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
1800 ggaaccgtaa aaaggccgcg ttgctggcgt ttttcgatag gctccgcccc
cctgacgagc 1860 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
gacaggacta taaagatacc 1920 aggcgtttcc ccctggaagc tccctcgtgc
gctctcctgt tccgaccctg ccgcttaccg 1980 gatacctgtc cgcctttctc
ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 2040 ggtatctcag
ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 2100
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
2160 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
aggtatgtag 2220 gcggtgctac agagttcttg aagtggtggc ctaactacgg
ctacactaga aggacagtat 2280 ttggtatctg cgctctgctg aagccagtta
ccttcggaaa aagagttggt agctcttgat 2340 ccggcaaaca aaccaccgct
ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 2400 gcagaaaaaa
aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 2460
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
2520 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
gagtaaactt 2580 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
ctcagcgatc tgtctatttc 2640 gttcatccat agttgcctga ctccccgtcg
tgtagataac tacgatacgg gagggcttac 2700 catctggccc cagtgctgca
atgataccgc gagacccacg ctcaccggct ccagatttat 2760 cagcaataaa
ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 2820
cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
2880 gtttgcgcaa cgttgttggc attgctacag gcatcgtggt gtcacgctcg
tcgtttggta 2940 tggcttcatt cagctccggt tcccaacgat caaggcgagt
tacatgatcc cccatgttgt 3000 gcaaaaaagc ggttagctcc ttcggtcctc
cgatcgttgt cagaagtaag ttggccgcag 3060 tgttatcact catggttatg
gcagcactgc ataattctct tactgtcatg ccatccgtaa 3120 gatgcttttc
tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 3180
gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
3240 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
atcttaccgc 3300 tgttgagatc cagttcgatg taacccactc gtgcacccaa
ctgatcttca gcatctttta 3360 ctttcaccag cgtttctggg tgagcaaaaa
caggaaggca aaatgccgca aaaaagggaa 3420 taagggcgac acggaaatgt
tgaatactca tactcttcct ttttcaatat tattgaagca 3480 tttatcaggg
ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 3540
aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
3600 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
ctcgcgcgtt 3660 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
ggagacggtc acagcttgtc 3720 tgtaagcgga tgccgggagc agacaagccc
gtcagggcgc gtcagcgggt gttggcgggt 3780 gtcggggctg gcttaactat
gcggcatcag agcagattgt actgagagtg caccatatgc 3840 ggtgtgaaat
accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat 3900
tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
3960 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
ttttcccagt 4020 cacgacgttg taaaacgacg gccagtgaat tggatttagg
tgacactata gaatacgaat 4080 tc 4082
* * * * *